USP 29 HPLC and Other

INTERPRETATION OF CHROMATOGRAMS

Figure 1 represents a typical chromatographic separation of two substances, 1 and 2, where t1 and t2 are the respective retention times, h, h/2, and Wh/2 are the height, the half-height, and the width at half-height, respectively, for peak 1. W1 and W2 are the respective widths of peaks 1 and 2 at the baseline. Air peaks are a feature of gas chromatograms and correspond to the solvent front in liquid chromatography.

Figure 1.Chromatographic separation of two substances

Chromatographic retention times are characteristic of the compounds they represent but are not unique. Coincidence of retention times of a test and a reference substance can be used as a feature in construction of an identity profile but is insufficient on its own to establish identity. Absolute retention times of a given compound vary from one chromatogram to the next. Comparisons are normally made in terms of relative retention, , which is calculated by the equation:

in which t2 and t1 are the retention times, measured from the point of injection, of the test and reference substances, respectively, determined under identical experimental conditions on the same column, and ta is the retention time of a nonretained substance, such as methane in the case of gas chromatography.

In this and the following expressions, the corresponding retention volumes or linear separations on the chromatogram, both of which are directly proportional to retention time, may be substituted in the equations. Where the value of ta is small, RR may be estimated from the retention times measured from the point of injection (t2/t1).

The number of theoretical plates, N, is a measure of column efficiency. For Gaussian peaks, it is calculated by the equation:

in which t is the retention time of the substance and W is the width of the peak at its base, obtained by extrapolating the relatively straight sides of the peak to the baseline. Wh/2 is the peak width at half-height, obtained directly by electronic integrators. The value of N depends upon the substance being chromatographed as well as the operating conditions such as mobile phase or carrier gas flow rates and temperature, the quality of the packing, the uniformity of the packing within the column and, for capillary columns, the thickness of the stationary phase film, and the internal diameter and length of the column.

The separation of two components in a mixture, the resolution, R, is determined by the equation:

in which t2 and t1 are the retention times of the two components, and W2 and W1 are the corresponding widths at the bases of the peaks obtained by extrapolating the relatively straight sides of the peaks to the baseline.

Where electronic integrators are used, it may be convenient to determine the resolution, R, by the equation:

and to determine the number of theoretical plates, N, by the equation:

however, in the event of dispute, only equations based on peak width at baseline are to be used.

Peak areas and peak heights are usually proportional to the quantity of compound eluting. These are commonly measured by electronic integrators but may be determined by more classical approaches. Peak areas are generally used but may be less accurate if peak interference occurs. For manual measurements, the chart should be run faster than usual, or a comparator should be used to measure the width at half-height and the width at the base of the peak, to minimize error in these measurements. For accurate quantitative work, the components to be measured should be separated from any interfering components. Peak tailing and fronting and the measurement of peaks on solvent tails are to be avoided.

Chromatographic purity tests for drug raw materials are sometimes based on the determination of peaks due to impurities, expressed as a percentage of the area due to the drug peak. It is preferable, however, to compare impurity peaks to the chromatogram of a standard at a similar concentration. The standard may be the drug itself at a level corresponding to, for example, 0.5% impurity, or in the case of toxic or signal impurities, a standard of the impurity itself.

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SYSTEM SUITABILITY

System suitability tests are an integral part of gas and liquid chromatographic methods. They are used to verify that the detection sensitivity,USP29 (Official June 1, 2006) resolution, and reproducibility of the chromatographic system are adequate for the analysis to be done. The tests are based on the concept that the equipment, electronics, analytical operations, and samples to be analyzed constitute an integral system that can be evaluated as such.

The detection sensitivity is a measure used to ensure the suitability of a given chromatographic procedure for the complete detection of the impurities in the Chromatographic purity or Related compounds tests by injecting a volume of a quantitation limit solution equal to that of the Test solution. Unless otherwise specified in the individual monograph, the quantitation limit solution may be prepared by dissolving the drug substance Reference Standard in the same solvent as that used for the Test solution at a 0.05% concentration level relative to the amount of drug substance in the Test solution for drug substances, and a 0.1% level relative to the amount of drug substance in the Test solution for drug products. The signal-to-noise ratio for the drug substance peak obtained with the quantitation limit solution should be not less than 10.USP29 (Official June 1, 2006)

The resolution, R, [NOTE—All terms and symbols are defined in the Glossary of Symbols] is a function of column efficiency, N, and is specified to ensure that closely eluting compounds are resolved from each other, to establish the general resolving power of the system, and to ensure that internal standards are resolved from the drug. Column efficiency may be specified also as a system suitability requirement, especially if there is only one peak of interest in the chromatogram; however, it is a less reliable means to ensure resolution than direct measurement. Column efficiency is a measure of peak sharpness, which is important for the detection of trace components.

Replicate injections of a standard preparation used in the assay or other standard solution are compared to ascertain whether requirements for precision are met. Unless otherwise specified in the individual monograph, data from five replicate injections of the analyte are used to calculate the relative standard deviation, SR, if the requirement is 2.0% or less; data from six replicate injections are used if the relative standard deviation requirement is more than 2.0%.

The tailing factor, T, a measure of peak symmetry, is unity for perfectly symmetrical peaks and its value increases as tailing becomes more pronounced (see Figure 2). In some cases, values less than unity may be observed. As peak asymmetry increases, integration, and hence precision, becomes less reliable.

Figure 2.Asymmetrical chromatographic peak

These tests are performed by collecting data from replicate injections of standard or other solutions as specified in the individual monograph. The specification of definitive parameters in a monograph does not preclude the use of other suitable operating conditions (see Procedures under Tests and Assays in the General Notices). Adjustments of operating conditions to meet system suitability requirements may be necessary.

Unless otherwise directed in the monograph, system suitability parameters are determined from the analyte peak.

To ascertain the effectiveness of the final operating system, it should be subjected to suitability testing. Replicate injections of the standard preparation required to demonstrate adequate system precision may be made before the injection of samples or may be interspersed among sample injections. System suitability must be demonstrated throughout the run by injection of an appropriate control preparation at appropriate intervals. The control preparation can be a standard preparation or a solution containing a known amount of analyte and any additional materials useful in the control of the analytical system, such as excipients or impurities. Whenever there is a significant change in equipment or in a critical reagent, suitability testing should be performed before the injection of samples. No sample analysis is acceptable unless the requirements of system suitability have been met. Sample analyses obtained while the system fails requirements are unacceptable.

GLOSSARY OF SYMBOLS

To promote uniformity of interpretation, the following symbols and definitions are employed where applicable in presenting formulas in the individual monographs. [NOTE—Where the terms W and t both appear in the same equation they must be expressed in the same units.]

		relative retention,
cR, cI, cU		concentrations of Reference Standard, internal standard, and analyte in a particular solution.
CA		concentration ratio of analyte and internal standard in test solution or Assay preparation,
CS		concentration ratio of Reference Standard and internal standard in Standard solution,
f		distance from the peak maximum to the leading edge of the peak, the distance being measured at a point 5% of the peak height from the baseline.
k¢		capacity factor,
N		number of theoretical plates in a chromatographic column,
qR, qI, qU		total quantities (weights) of Reference Standard, internal standard, and analyte in a particular solution.
QA		quantity ratio of analyte and internal standard in test solution or Assay preparation,
QS		quantity ratio of Reference Standard and internal standard in Standard solution,
rS		peak response of the Reference Standard obtained from a chromatogram.
rU		peak response of the analyte obtained from a chromatogram.
R		resolution between two chromatographic peaks,
RF		chromatographic retardation factor equal to the ratio of the distance from the origin to the center of a zone divided by the distance from the origin to the solvent front.
RR		relative retention
RR		relative retention time
RS		peak response ratio for Standard preparation containing Reference Standard and internal standard,
RU		peak response ratio for Assay preparation containing the analyte and internal standard,
SR (%)		relative standard deviation in percentage,
		where Xi is an individual measurement in a set of N measurements and X is the arithmetic mean of the set.
T		tailing factor,
t		retention time measured from time of injection to time of elution of peak maximum.
ta		retention time of nonretarded component, air with thermal conductivity detection.
W		width of peak measured by extrapolating the relatively straight sides to the baseline.
Wh/2		width of peak at half height.
W0.05		width of peak at 5% height.

641 COMPLETENESS OF SOLUTION

Place the quantity of the substance specified in the individual monograph in a meticulously cleansed, glass-stoppered, 10-mL glass cylinder approximately 13 mm × 125 mm in size. Using the solvent that is specified in the monograph or on the label of the product, fill the cylinder almost to the constriction at the neck. Shake gently to effect solution: the solution is not less clear than an equal volume of the same solvent contained in a similar vessel and examined similarly.

643 TOTAL ORGANIC CARBON

Total organic carbon (TOC) is an indirect measure of organic molecules present in pharmaceutical waters measured as carbon. Organic molecules are introduced into the water from the source water, from purification and distribution system materials, and from biofilm growing in the system. TOC can also be used as a process control attribute to monitor the performance of unit operations comprising the purification and distribution system.

A number of acceptable methods exist for analyzing TOC. This chapter does not limit or prevent alternative technologies from being used, but provides guidance on how to qualify these analytical technologies for use as well as guidance on how to interpret instrument results for use as a limit test. The Standard Solution is a theoretically easy-to-oxidize solution that gives an instrument response at the attribute limit. The analytical technology is qualified by challenging the capability of the instrument using a theoretically difficult to oxidize solution in the system suitability portion of the method.

Analytical technologies utilized to measure TOC share the objective of completely oxidizing the organic molecules in an aliquot of sample water to carbon dioxide (CO2), measuring the resultant CO2 levels, and expressing this response as carbon concentration. All technologies must discriminate between the inorganic carbon, which may be present in the water from sources such as dissolved CO2 and bicarbonate, and the CO2 generated from the oxidation of organic molecules in the sample.

Two general approaches are used to measure TOC. One approach determines TOC by subtracting the measured inorganic carbon (IC) from the measured total carbon (TC), which is the sum of organic carbon and inorganic carbon:

TOC = TC – IC.

The other approach first purges the IC from the sample before any carbon measurement is performed. However, this IC purging step also purges some of the organic molecules, which can be retrapped, oxidized to CO2, and quantitated as purgeable organic carbon (POC). The remaining organic matter in the sample is also oxidized to CO2 and quantitated as nonpurgeable organic carbon (NPOC). In this approach, TOC is the sum of POC and NPOC:

TOC = POC + NPOC.

In pharmaceutical waters, the amount of POC is negligible and can be discounted. Therefore, for the purpose of this methodology, NPOC is equivalent to TOC.

Apparatus Requirements— This test method is performed either as an on-line test or as an off-line laboratory test using a calibrated instrument. The suitability of the apparatus must be periodically demonstrated as described below. In addition, it must have a manufacturer's specified limit of detection of 0.05 mg of carbon per L (0.05 ppm of carbon) or lower.

USP Reference Standards 11— USP 1,4-Benzoquinone RS. USP Sucrose RS.

Reagent Water— Use water having a TOC level of not more than 0.10 mg per L. [NOTE—A conductivity requirement may be necessary to ensure method reliability.]

Glassware Preparation— Organic contamination of glassware results in higher TOC values. Therefore, use glassware and sample containers that have been scrupulously cleaned of organic residues. Any method that is effective in removing organic matter can be used (see Cleaning Glass Apparatus 1051). Use Reagent Water for the final rinse.

Standard Solution— Unless otherwise directed in the individual monograph, dissolve in the Reagent Water an accurately weighed quantity of USP Sucrose RS, to obtain a solution having a concentration of about 1.2 mg of sucrose per L (0.50 mg of carbon per liter).

Test Solution— [NOTE—Use extreme caution when obtaining samples for TOC analysis. Water samples can be easily contaminated during the process of sampling and transportation to a testing facility.] Collect the Test Solution in a tight container with minimal head space, and test in a timely manner to minimize the impact of organic contamination from the closure and container.

System Suitability Solution— Dissolve in Reagent Water an accurately weighed quantity of USP 1,4-Benzoquinone RS to obtain a solution having a concentration of 0.75 mg per L (0.50 mg of carbon per liter).

Reagent Water Control— Use a suitable quantity of Reagent Water obtained at the same time as that used in the preparation of the Standard Solution and the System Suitability Solution.

Other Control Solutions— Prepare appropriate reagent blank solutions or other specified solutions needed for establishing the apparatus baseline or for calibration adjustments following the manufacturer's instructions, and run the appropriate blanks to zero the instrument.

System Suitability— Test the Reagent Water Control in the apparatus, and record the response, rw. Repeat the test using the Standard Solution, and record the response, rS. Calculate the corrected Standard Solution response, which is also the limit response, by subtracting the Reagent Water Control response from the response of the Standard Solution. The theoretical limit of 0.50 mg of carbon per L is equal to the corrected Standard Solution response, rS – rw. Test the System Suitability Solution in the apparatus, and record the response, rss. Calculate the corrected System Suitability Solution response by subtracting the Reagent Water Control response from the response of the System Suitability Solution, rss – rw. Calculate the response efficiency for the System Suitability Solution by the formula:

100[(rss – rw) / (rS – rw)].

The system is suitable if the response efficiency is not less than 85% and not more than 115% of the theoretical response.

Procedure— Perform the test on the Test Solution, and record the response, rU. The Test Solution meets the requirements if rU is not more than the limit response, rS – rw. This method also can be performed alternatively using on-line instrumentation that has been appropriately calibrated, standardized, and has demonstrated acceptable system suitability. The acceptability of such on-line instrumentation for quality attribute testing is dependent on its location(s) in the water system. These instrument location(s) and responses must reflect the quality of the water used.

645 WATER CONDUCTIVITY

Electrical conductivity in water is a measure of the ion-facilitated electron flow through it. Water molecules dissociate into ions as a function of pH and temperature and result in a very predictable conductivity. Some gases, most notably carbon dioxide, readily dissolve in water and interact to form ions, which predictably affect conductivity as well as pH. For the purpose of this discussion, these ions and their resulting conductivity can be considered intrinsic to the water.

Water conductivity is also affected by the presence of extraneous ions. The extraneous ions used in modeling the conductivity specifications described below are the chloride and sodium ions. The conductivity of the ubiquitous chloride ion (at the theoretical endpoint concentration of 0.47 ppm when it was a required attribute test in USP XXII and earlier revisions) and the ammonium ion at the limit of 0.3 ppm represents a major portion of the allowed water impurity level. A balancing quantity of cations, such as sodium ion, is included in this allowed impurity level to maintain electroneutrality. Extraneous ions such as these may have significant impact on the water's chemical purity and suitability for use in pharmaceutical applications. The combined conductivities of the intrinsic and extraneous ions vary as a function of pH and are the basis for the conductivity specifications described in the accompanying table and used when performing Stage 3 of the test method. Two preliminary stages are included in the test method. If the test conditions and conductivity limits are met at either of these preliminary stages, the water meets the requirements of this test. Proceeding to the third stage of the test in these circumstances is unnecessary. Only in the event of failure at the final test stage is the sample judged noncompliant with the requirements of the test.

INSTRUMENT SPECIFICATIONS AND OPERATING PARAMETERS

Water conductivity must be measured accurately using calibrated instrumentation. The conductivity cell constant, a factor used as a multiplier for the scale reading from the meter, must be known within ±2%. The cell constant can be verified directly by using a solution of known conductivity, or indirectly by comparing the instrument reading taken with the cell in question to readings from a cell of known or certified cell constant.

Meter calibration is accomplished by replacing the conductivity cell with NIST-traceable precision resistors (accurate to ±0.1% of the stated value) or an equivalently accurate adjustable resistance device, such as a Wheatstone Bridge, to give a predicted instrument response. Each scale on the meter may require separate calibration prior to use. The frequency of recalibration is a function of instrument design, degree of use, etc. However, because some multiple-scale instruments have a single calibration adjustment, recalibration may be required between each use of a different scale. The instrument must have a minimum resolution of 0.1 µS/cm* on the lowest range. Excluding the cell accuracy, the instrument accuracy must be ±0.1 µS/cm.

Because temperature has a substantial impact on conductivity readings of specimens at high and low temperatures, many instruments automatically correct the actual reading to display the value that theoretically would be observed at the nominal temperature of 25. This is done using a temperature sensor in the conductivity cell probe and an algorithm in the instrument's circuitry. This temperature compensation algorithm may not be accurate. Conductivity values used in this method are nontemperature-compensated measurements. Accuracy of the temperature measurement must be ±2.

The procedure described below is designed for measuring the conductivity of Purified Water and Water for Injection. Stage 1 of the procedure below may alternatively be performed (with the appropriate modifications to Step 1) using on-line instrumentation that has been appropriately calibrated, whose cell constants have been accurately determined, and whose temperature compensation function has been disabled. The suitability of such on-line instrumentation for quality control testing is also dependent on its location(s) in the water system. The selected instrument location(s) must reflect the quality of the water used.

PROCEDURE

Stage 1

1. Determine the temperature of the water and the conductivity of the water using a nontemperature-compensated conductivity reading. The measurement may be performed in a suitable container or as an on-line measurement.

2. Using the Stage 1—Temperature and Conductivity Requirements table, find the temperature value that is not greater than the measured temperature, i.e., the next lower temperature. The corresponding conductivity value on this table is the limit. [NOTE—Do not interpolate.]

3. If the measured conductivity is not greater than the table value, the water meets the requirements of the test for conductivity. If the conductivity is higher than the table value, proceed with Stage 2.

Stage 1—Temperature and Conductivity Requirements
(for nontemperature-compensated conductivity measurements only)

Temperature	Conductivity Requirement (µS/cm)
0	0.6
5	0.8
10	0.9
15	1.0
20	1.1
25	1.3
30	1.4
35	1.5
40	1.7
45	1.8
50	1.9
55	2.1
60	2.2
65	2.4
70	2.5
75	2.7
80	2.7
85	2.7
90	2.7
95	2.9
100	3.1

Stage 2

4. Transfer a sufficient amount of water (100 mL or more) to a suitable container, and stir the test specimen. Adjust the temperature, if necessary, and, while maintaining it at 25 ± 1, begin vigorously agitating the test specimen while periodically observing the conductivity. When the change in conductivity (due to uptake of atmospheric carbon dioxide) is less than a net of 0.1 µS/cm per 5 minutes, note the conductivity.

5. If the conductivity is not greater than 2.1 µS/cm, the water meets the requirements of the test for conductivity. If the conductivity is greater than 2.1 µS/cm, proceed with Stage 3.

Stage 3

6. Perform this test within approximately 5 minutes of the conductivity determination in Step 5, while maintaining the sample temperature at 25 ± 1. Add a saturated potassium chloride solution to the same water sample (0.3 mL per 100 mL of the test specimen), and determine the pH to the nearest 0.1 pH unit, as directed under pH 791.

7. Referring to the Stage 3—pH and Conductivity Requirements table, determine the conductivity limit at the measured pH value. If the measured conductivity in Step 4 is not greater than the conductivity requirements for the pH determined in Step 6, the water meets the requirements of the test for conductivity. If either the measured conductivity is greater than this value or the pH is outside the range of 5.0 to 7.0, the water does not meet the requirements of the test for conductivity.

Stage 3—pH and Conductivity Requirements
(for atmosphere- and temperature-equilibrated samples only)

pH	Conductivity Requirement (µS/cm)
5.0	4.7
5.1	4.1
5.2	3.6
5.3	3.3
5.4	3.0
5.5	2.8
5.6	2.6
5.7	2.5
5.8	2.4
5.9	2.4
6.0	2.4
6.1	2.4
6.2	2.5
6.3	2.4
6.4	2.3
6.5	2.2
6.6	2.1
6.7	2.6
6.8	3.1
6.9	3.8
7.0	4.6

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* µS/cm (microsiemens per centimeter) = µmho/cm = reciprocal of megohm-cm.

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701 DISINTEGRATION

This general chapter is harmonized with the corresponding texts of the European Pharmacopoeia and/or the Japanese Pharmacopoeia. The texts of these pharmacopeias are therefore interchangeable, and the methods of the European Pharmacopoeia and/or the Japanese Pharmacopoeia may be used for demonstration of compliance instead of the present general chapter. These pharmacopeias have undertaken not to make any unilateral change to this harmonized chapter.

Portions of the present general chapter text that are national USP text, and therefore not part of the harmonized text, are marked with symbols () to specify this fact.

This test is provided to determine whether tablets or capsules disintegrate within the prescribed time when placed in a liquid medium at the experimental conditions presented below. Compliance with the limits on Disintegration stated in the individual monographs is required except where the label states that the tablets or capsules are intended for use as troches, or are to be chewed, or are designed as extended-release dosage forms or delayed-release dosage forms. Determine the type of units under test from the labeling and from observation, and apply the appropriate procedure to 6 or more dosage units.

For the purposes of this test, disintegration does not imply complete solution of the unit or even of its active constituent. Complete disintegration is defined as that state in which any residue of the unit, except fragments of insoluble coating or capsule shell, remaining on the screen of the test apparatus or adhering to the lower surface of the disk, if used, is a soft mass having no palpably firm core.

APPARATUS

The apparatus consists of a basket-rack assembly, a 1000-mL, low-form beaker, 138 to 160 mm in height and having an inside diameter of 97 to 115 mm for the immersion fluid, a thermostatic arrangement for heating the fluid between 35 and 39, and a device for raising and lowering the basket in the immersion fluid at a constant frequency rate between 29 and 32 cycles per minute through a distance of not less than 53 mm and not more than 57 mm. The volume of the fluid in the vessel is such that at the highest point of the upward stroke the wire mesh remains at least 15 mm below the surface of the fluid and descends to not less than 25 mm from the bottom of the vessel on the downward stroke. At no time should the top of the basket-rack assembly become submerged. The time required for the upward stroke is equal to the time required for the downward stroke, and the change in stroke direction is a smooth transition, rather than an abrupt reversal of motion. The basket-rack assembly moves vertically along its axis. There is no appreciable horizontal motion or movement of the axis from the vertical.

Basket-Rack Assembly— The basket-rack assembly consists of six open-ended transparent tubes, each 77.5 ± 2.5 mm long and having an inside diameter of 20.7 to 23 mm and a wall 1.0 to 2.8 mm thick; the tubes are held in a vertical position by two plates, each 88 to 92 mm in diameter and 5 to 8.5 mm in thickness, with six holes, each 22 to 26 mm in diameter, equidistant from the center of the plate and equally spaced from one another. Attached to the under surface of the lower plate is a woven stainless steel wire cloth, which has a plain square weave with 1.8- to 2.2-mm apertures and with a wire diameter of 0.57 to 0.66 mm. The parts of the apparatus are assembled and rigidly held by means of three bolts passing through the two plates. A suitable means is provided to suspend the basket-rack assembly from the raising and lowering device using a point on its axis.

The design of the basket-rack assembly may be varied somewhat, provided the specifications for the glass tubes and the screen mesh size are maintained. The basket-rack assembly conforms to the dimensions found in Figure 1.

Disks— The use of disks is permitted only where specified or allowed in the monograph. If specified in the individual monograph, each tube is provided with a cylindrical disk 9.5 ± 0.15 mm thick and 20.7 ± 0.15 mm in diameter. The disk is made of a suitable transparent plastic material having a specific gravity of between 1.18 and 1.20. Five parallel 2 ± 0.1-mm holes extend between the ends of the cylinder. One of the holes is centered on the cylindrical axis. The other holes are centered 6 ± 0.2 mm from the axis on imaginary lines perpendicular to the axis and parallel to each other. Four identical trapezoidal-shaped planes are cut into the wall of the cylinder, nearly perpendicular to the ends of the cylinder. The trapezoidal shape is symmetrical; its parallel sides coincide with the ends of the cylinder and are parallel to an imaginary line connecting the centers of two adjacent holes 6 mm from the cylindrical axis. The parallel side of the trapezoid on the bottom of the cylinder has a length of 1.6 ± 0.1 mm, and its bottom edges lie at a depth of 1.6 ± 0.1 mm from the cylinder's circumference. The parallel side of the trapezoid on the top of the cylinder has a length of 9.4 ± 0.2 mm, and its center lies at a depth of 2.6 ± 0.1 mm from the cylinder's circumference. All surfaces of the disk are smooth. If the use of disks is specified in the individual monograph, add a disk to each tube, and operate the apparatus as directed under Procedure. The disks conform to dimensions found in Figure 11.

Fig. 1. Disintegration apparatus. (All dimensions are expressed in mm.)

PROCEDURE

Uncoated Tablets— Place 1 dosage unit in each of the six tubes of the basket and, if prescribed, add a disk. Operate the apparatus, using water or the specified medium as the immersion fluid, maintained at 37 ± 2. At the end of the time limit specified in the monograph, lift the basket from the fluid, and observe the tablets: all of the tablets have disintegrated completely. If 1 or 2 tablets fail to disintegrate completely, repeat the test on 12 additional tablets. The requirement is met if not fewer than 16 of the total of 18 tablets tested are disintegrated.

Plain-Coated Tablets— Apply the test for Uncoated Tablets, operating the apparatus for the time specified in the individual monograph.

Delayed-Release (Enteric-Coated) Tablets— Place 1 tablet in each of the six tubes of the basket and, if the tablet has a soluble external sugar coating, immerse the basket in water at room temperature for 5 minutes. Then operate the apparatus using simulated gastric fluid TS maintained at 37 ± 2 as the immersion fluid. After 1 hour of operation in simulated gastric fluid TS, lift the basket from the fluid, and observe the tablets: the tablets show no evidence of disintegration, cracking, or softening. Operate the apparatus, using simulated intestinal fluid TS maintained at 37 ± 2 as the immersion fluid, for the time specified in the monograph. Lift the basket from the fluid, and observe the tablets: all of the tablets disintegrate completely. If 1 or 2 tablets fail to disintegrate completely, repeat the test on 12 additional tablets: not fewer than 16 of the total of 18 tablets tested disintegrate completely.

Buccal Tablets— Apply the test for Uncoated Tablets. After 4 hours, lift the basket from the fluid, and observe the tablets: all of the tablets have disintegrated. If 1 or 2 tablets fail to disintegrate completely, repeat the test on 12 additional tablets: not fewer than 16 of the total of 18 tablets tested disintegrate completely.

Sublingual Tablets— Apply the test for Uncoated Tablets. At the end of the time limit specified in the individual monograph: all of the tablets have disintegrated. If 1 or 2 tablets fail to disintegrate completely, repeat the test on 12 additional tablets: not fewer than 16 of the total of 18 tablets tested disintegrate completely.

Hard Gelatin Capsules— Apply the test for Uncoated Tablets. Attach a removable wire cloth, which has a plain square weave with 1.8- to 2.2-mm mesh apertures and with a wire diameter of 0.60 to 0.655 mm, as described under Basket-Rack Assembly, to the surface of the upper plate of the basket-rack assembly. Observe the capsules within the time limit specified in the individual monograph: all of the capsules have disintegrated except for fragments from the capsule shell. If 1 or 2 capsules fail to disintegrate completely, repeat the test on 12 additional capsules: not fewer than 16 of the total of 18 capsules tested disintegrate completely.

Soft Gelatin Capsules— Proceed as directed under Hard Gelatin Capsules.

(Official April 1, 2006)

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1 The use of automatic detection employing modified disks is permitted where the use of disks is specified or allowed. Such disks must comply with the requirements for density and dimension given in this chapter.

711 DISSOLUTION

This general chapter is harmonized with the corresponding texts of the European Pharmacopoeia and/or the Japanese Pharmacopoeia. The texts of these pharmacopeias are therefore interchangeable, and the methods of the European Pharmacopoeia or the Japanese Pharmacopoeia may be used for demonstration of compliance instead of the present general chapter. These pharmacopeias have undertaken not to make any unilateral change to this harmonized chapter.

Portions of the present general chapter text that are national USP text, and therefore not part of the harmonized text, are marked with symbols () to specify this fact.

This test is provided to determine compliance with the dissolution requirements where stated in the individual monograph for dosage forms administered orally. In this general chapter, a dosage unit is defined as 1 tablet or 1 capsule or the amount specified. Of the types of apparatus described herein, use the one specified in the individual monograph. Where the label states that an article is enteric-coated, and where a dissolution or disintegration test that does not specifically state that it is to be applied to delayed-release articles is included in the individual monograph, the procedure and interpretation given for Delayed-Release Dosage Forms is applied unless otherwise specified in the individual monograph. For hard or soft gelatin capsules and gelatin-coated tablets that do not conform to the Dissolution specification, repeat the test as follows. Where water or a medium with a pH of less than 6.8 is specified as the Medium in the individual monograph, the same Medium specified may be used with the addition of purified pepsin that results in an activity of 750,000 Units or less per 1000 mL. For media with a pH of 6.8 or greater, pancreatin can be added to produce not more than 1750 USP Units of protease activity per 1000 mL.

USP Reference Standards 11— USP Chlorpheniramine Maleate Extended-Release Tablets RS (Drug Release Calibrator, Single Unit). USP Prednisone Tablets RS (Dissolution Calibrator, Disintegrating). USP Salicylic Acid Tablets RS. (Dissolution Calibrator, Nondisintegrating).

APPARATUS

Apparatus 1 (Basket Apparatus)

The assembly consists of the following: a vessel, which may be covered, made of glass or other inert, transparent material1 ; a motor; a metallic drive shaft; and a cylindrical basket. The vessel is partially immersed in a suitable water bath of any convenient size or heated by a suitable device such as a heating jacket. The water bath or heating device permits holding the temperature inside the vessel at 37 ± 0.5 during the test and keeping the bath fluid in constant, smooth motion. No part of the assembly, including the environment in which the assembly is placed, contributes significant motion, agitation, or vibration beyond that due to the smoothly rotating stirring element. An apparatus that permits observation of the specimen and stirring element during the test is preferable. The vessel is cylindrical, with a hemispherical bottom and with one of the following dimensions and capacities: for a nominal capacity of 1 L, the height is 160 mm to 210 mm and its inside diameter is 98 mm to 106 mm; for a nominal capacity of 2 L, the height is 280 mm to 300 mm and its inside diameter is 98 mm to 106 mm; and for a nominal capacity of 4 L, the height is 280 mm to 300 mm and its inside diameter is 145 mm to 155 mm. Its sides are flanged at the top. A fitted cover may be used to retard evaporation.2 The shaft is positioned so that its axis is not more than 2 mm at any point from the vertical axis of the vessel and rotates smoothly and without significant wobble that could affect the results. A speed-regulating device is used that allows the shaft rotation speed to be selected and maintained at the specified rate given in the individual monograph, within ±4%.

Shaft and basket components of the stirring element are fabricated of stainless steel, type 316, or other inert material, to the specifications shown in Figure 1. A basket having a gold coating of about 0.0001 inch (2.5 µm) thick may be used. A dosage unit is placed in a dry basket at the beginning of each test. The distance between the inside bottom of the vessel and the bottom of the basket is maintained at 25 ± 2 mm during the test.

Fig. 1. Basket Stirring Element

Apparatus 2 (Paddle Apparatus)

Use the assembly from Apparatus 1, except that a paddle formed from a blade and a shaft is used as the stirring element. The shaft is positioned so that its axis is not more than 2 mm from the vertical axis of the vessel at any point and rotates smoothly without significant wobble that could affect the results. The vertical center line of the blade passes through the axis of the shaft so that the bottom of the blade is flush with the bottom of the shaft. The paddle conforms to the specifications shown in Figure 2. The distance of 25 ± 2 mm between the bottom of the blade and the inside bottom of the vessel is maintained during the test. The metallic or suitably inert, rigid blade and shaft comprise a single entity. A suitable two-part detachable design may be used provided the assembly remains firmly engaged during the test. The paddle blade and shaft may be coated with a suitable coating so as to make them inert. The dosage unit is allowed to sink to the bottom of the vessel before rotation of the blade is started. A small, loose piece of nonreactive material, such as not more than a few turns of wire helix, may be attached to dosage units that would otherwise float. An alternative sinker device is shown in Figure 2a. Other validated sinker devices may be used.

Fig. 2. Paddle Stirring Element

Fig. 2a. Alternative sinker. All dimensions are expressed in mm.

Apparatus 3 (Reciprocating Cylinder)

NOT ACCEPTED BY THE JAPANESE PHARMACOPOEIA

The assembly consists of a set of cylindrical, flat-bottomed glass vessels; a set of glass reciprocating cylinders; inert fittings (stainless steel type 316 or other suitable material), and screens that are made of suitable nonsorbing and nonreactive material and that are designed to fit the tops and bottoms of the reciprocating cylinders; and a motor and drive assembly to reciprocate the cylinders vertically inside the vessels and, if desired, index the reciprocating cylinders horizontally to a different row of vessels. The vessels are partially immersed in a suitable water bath of any convenient size that permits holding the temperature at 37 ± 0.5 during the test. No part of the assembly, including the environment in which the assembly is placed, contributes significant motion, agitation, or vibration beyond that due to the smooth, vertically reciprocating cylinder. A device is used that allows the reciprocation rate to be selected and maintained at the specified dip rate given in the individual monograph within ±5%. An apparatus that permits observation of the specimens and reciprocating cylinders is preferable. The vessels are provided with an evaporation cap that remains in place for the duration of the test. The components conform to the dimensions shown in Figure 3 unless otherwise specified in the individual monograph.

Fig. 3. Apparatus 3 (reciprocating cylinder)

Apparatus 4 (Flow-Through Cell)

The assembly consists of a reservoir and a pump for the Dissolution Medium; a flow-through cell; and a water bath that maintains the Dissolution Medium at 37 ± 0.5. Use the specified cell size as given in the individual monograph.

The pump forces the Dissolution Medium upwards through the flow-through cell. The pump has a delivery range between 240 and 960 mL per hour, with standard flow rates of 4, 8, and 16 mL per minute. It must deliver a constant flow (±5% of the nominal flow rate); the flow profile is sinusoidal with a pulsation of 120 ± 10 pulses per minute.

The flow-through cell (see Figures 4 and 5), of transparent and inert material, is mounted vertically with a filter system (specified in the individual monograph) that prevents escape of undissolved particles from the top of the cell; standard cell diameters are 12 and 22.6 mm; the bottom cone is usually filled with small glass beads of about 1-mm diameter with one bead of about 5 mm positioned at the apex to protect the fluid entry tube; and a tablet holder (see Figures 4 and 5) is available for positioning of special dosage forms, for example, inlay tablets. The cell is immersed in a water bath, and the temperature is maintained at 37 ± 0.5.

Fig. 4. Large cell for tablets and capsules (top) Tablet holder for the large cell (bottom) (All measurements are expressed in mm unless noted otherwise.)

Fig. 5. Small cell for tablets and capsules (top) Tablet holder for the small cell (bottom) (All measurements are expressed in mm unless noted otherwise.)

The apparatus uses a clamp mechanism and two O-rings to assemble the cell. The pump is separated from the dissolution unit in order to shield the latter against any vibrations originating from the pump. The position of the pump should not be on a level higher than the reservoir flasks. Tube connections are as short as possible. Use suitably inert tubing, such as polytef, with about 1.6-mm inner diameter and chemically inert flanged-end connections.

Apparatus Suitability

The determination of suitability of a test assembly to perform dissolution testing must include conformance to the dimensions and tolerances of the apparatus as given above. In addition, critical test parameters that have to be monitored periodically during use include volume and temperature of the Dissolution Medium, rotation speed (Apparatus 1 and Apparatus 2), dip rate (Apparatus 3), and flow rate of medium (Apparatus 4).

Determine the acceptable performance of the dissolution test assembly periodically. The suitability for the individual apparatus is demonstrated by the Apparatus Suitability Test.

Apparatus Suitability Test, Apparatus 1 and 2— Individually test 1 tablet of the USP Dissolution Calibrator, Disintegrating Type and 1 tablet of USP Dissolution Calibrator, Nondisintegrating Type, according to the operating conditions specified. The apparatus is suitable if the results obtained are within the acceptable range stated in the certificate for that calibrator in the apparatus tested.

Apparatus Suitability Test, Apparatus 3— Individually test 1 tablet of the USP Drug Release Tablets (Single Unit) according to the operating conditions specified. The apparatus is suitable if the results obtained are within the acceptable range stated in the certificate.

Apparatus Suitability Test, Apparatus 4— [To come.]

PROCEDURE

Apparatus 1 and Apparatus 2

Immediate-Release Dosage Forms

Place the stated volume of the Dissolution Medium (±1%) in the vessel of the specified apparatus given in the individual monograph, assemble the apparatus, equilibrate the Dissolution Medium to 37 ± 0.5, and remove the thermometer. Place 1 dosage unit in the apparatus, taking care to exclude air bubbles from the surface of the dosage unit, and immediately operate the apparatus at the specified rate given in the individual monograph. Within the time interval specified, or at each of the times stated, withdraw a specimen from a zone midway between the surface of the Dissolution Medium and the top of the rotating basket or blade, not less than 1 cm from the vessel wall. [NOTE—Where multiple sampling times are specified, replace the aliquots withdrawn for analysis with equal volumes of fresh Dissolution Medium at 37 or, where it can be shown that replacement of the medium is not necessary, correct for the volume change in the calculation. Keep the vessel covered for the duration of the test, and verify the temperature of the mixture under test at suitable times.] Perform the analysis as directed in the individual monograph using a suitable assay method.3 Repeat the test with additional dosage form units.

If automated equipment is used for sampling or the apparatus is otherwise modified, verification that the modified apparatus will produce results equivalent to those obtained with the standard apparatus described in this general chapter is necessary.

Dissolution Medium— A suitable dissolution medium is used. Use the solvent specified in the individual monograph. The volume specified refers to measurements made between 20 and 25. If the Dissolution Medium is a buffered solution, adjust the solution so that its pH is within 0.05 unit of the specified pH given in the individual monograph. [NOTE—Dissolved gases can cause bubbles to form, which may change the results of the test. If dissolved gases influence the dissolution results, dissolved gases should be removed prior to testing.4 ]

Time— Where a single time specification is given, the test may be concluded in a shorter period if the requirement for minimum amount dissolved is met. Specimens are to be withdrawn only at the stated times within a tolerance of ±2%.

Extended-Release Dosage Forms

Proceed as directed for Immediate-Release Dosage Forms.

Dissolution Medium— Proceed as directed for Immediate-Release Dosage Forms.

Time— The test-time points, generally three, are expressed in hours.

Delayed-Release Dosage Forms

NOT ACCEPTED BY THE JAPANESE PHARMACOPOEIA

Use Method A or Method B and the apparatus specified in the individual monograph. All test times stated are to be observed within a tolerance of ±2%, unless otherwise specified.

Method A—

Procedure (unless otherwise directed in the individual monograph)—

ACID STAGE— Place 750 mL of 0.1 N hydrochloric acid in the vessel, and assemble the apparatus. Allow the medium to equilibrate to a temperature of 37 ± 0.5. Place 1 dosage unit in the apparatus, cover the vessel, and operate the apparatus at the specified rate given in the monograph.

After 2 hours of operation in 0.1 N hydrochloric acid, withdraw an aliquot of the fluid, and proceed immediately as directed under Buffer Stage.

Perform an analysis of the aliquot using a suitable assay method. The procedure is specified in the individual monograph.

BUFFER STAGE— [NOTE—Complete the operations of adding the buffer and adjusting the pH within 5 minutes.]

With the apparatus operating at the rate specified in the monograph, add to the fluid in the vessel 250 mL of 0.20 M tribasic sodium phosphate that has been equilibrated to 37 ± 0.5. Adjust, if necessary, with 2 N hydrochloric acid or 2 N sodium hydroxide to a pH of 6.8 ± 0.05. Continue to operate the apparatus for 45 minutes, or for the specified time given in the individual monograph. At the end of the time period, withdraw an aliquot of the fluid, and perform the analysis using a suitable assay method. The procedure is specified in the individual monograph. The test may be concluded in a shorter time period than that specified for the Buffer Stage if the requirement for the minimum amount dissolved is met at an earlier time.

Method B—

Procedure (unless otherwise directed in the individual monograph)—

ACID STAGE— Place 1000 mL of 0.1 N hydrochloric acid in the vessel, and assemble the apparatus. Allow the medium to equilibrate to a temperature of 37 ± 0.5. Place 1 dosage unit in the apparatus, cover the vessel, and operate the apparatus at the rate specified in the monograph. After 2 hours of operation in 0.1 N hydrochloric acid, withdraw an aliquot of the fluid, and proceed immediately as directed under Buffer Stage.

Perform an analysis of the aliquot using a suitable assay method. The procedure is specified in the individual monograph.

BUFFER STAGE— [NOTE—For this stage of the procedure, use buffer that previously has been equilibrated to a temperature of 37 ± 0.5.] Drain the acid from the vessel, and add to the vessel 1000 mL of pH 6.8 phosphate buffer, prepared by mixing 0.1 N hydrochloric acid with 0.20 M tribasic sodium phosphate (3:1) and adjusting, if necessary, with 2 N hydrochloric acid or 2 N sodium hydroxide to a pH of 6.8 ± 0.05. [NOTE—This may also be accomplished by removing from the apparatus the vessel containing the acid and replacing it with another vessel containing the buffer and transferring the dosage unit to the vessel containing the buffer.]

Continue to operate the apparatus for 45 minutes, or for the specified time given in the individual monograph. At the end of the time period, withdraw an aliquot of the fluid, and perform the analysis using a suitable assay method. The procedure is specified in the individual monograph. The test may be concluded in a shorter time period than that specified for the Buffer Stage if the requirement for minimum amount dissolved is met at an earlier time.

Apparatus 3 (Reciprocating Cylinder)

NOT ACCEPTED BY THE JAPANESE PHARMACOPOEIA

Immediate-Release Dosage Forms

Place the stated volume of the Dissolution Medium in each vessel of the apparatus, assemble the apparatus, equilibrate the Dissolution Medium to 37 ± 0.5, and remove the thermometer. Place 1 dosage-form unit in each of the six reciprocating cylinders, taking care to exclude air bubbles from the surface of each dosage unit, and immediately operate the apparatus as specified in the individual monograph. During the upward and downward stroke, the reciprocating cylinder moves through a total distance of 9.9 to 10.1 cm. Within the time interval specified, or at each of the times stated, raise the reciprocating cylinders and withdraw a portion of the solution under test from a zone midway between the surface of the Dissolution Medium and the bottom of each vessel. Perform the analysis as directed in the individual monograph. If necessary, repeat the test with additional dosage-form units.

Dissolution Medium —Proceed as directed for Immediate-Release Dosage Forms under Apparatus 1 and Apparatus 2.

Time —Proceed as directed for Immediate-Release Dosage Forms under Apparatus 1 and Apparatus 2.

Extended-Release Dosage Forms

Proceed as directed for Immediate-Release Dosage Forms under Apparatus 3.

Dissolution Medium

—Proceed as directed for Extended-Release Dosage Forms under Apparatus 1 and Apparatus 2.

Time —Proceed as directed for Extended-Release Dosage Forms under Apparatus 1 and Apparatus 2.

Delayed-Release Dosage Forms

Proceed as described for Delayed-Release Dosage Forms, Method B under Apparatus 1 and Apparatus 2 using one row of vessels for the acid stage media and the following row of vessels for the buffer stage media and using the volume of medium specified (usually 300 mL).

Time —Proceed as directed for Immediate-Release Dosage Forms under Apparatus 1 and Apparatus 2.

Apparatus 4 (Flow-Through Cell)

Immediate-Release Dosage Forms

Place the glass beads into the cell specified in the monograph. Place 1 dosage unit on top of the beads or, if specified in the monograph, on a wire carrier. Assemble the filter head, and fix the parts together by means of a suitable clamping device. Introduce by the pump the Dissolution Medium warmed to 37 ± 0.5 through the bottom of the cell to obtain the flow rate specified in the individual monograph and measured with an accuracy of 5%. Collect the eluate by fractions at each of the times stated. Perform the analysis as directed in the individual monograph. Repeat the test with additional dosage-form units.

Dissolution Medium —Proceed as directed for Immediate-Release Dosage Forms under Apparatus 1 and Apparatus 2.

Time —Proceed as directed for Immediate-Release Dosage Forms under Apparatus 1 and Apparatus 2.

Extended-Release Dosage Forms

Proceed as directed for Immediate-Release Dosage Forms under Apparatus 4.

Dissolution Medium —Proceed as directed for Immediate-Release Dosage Forms under Apparatus 4.

Time —Proceed as directed for Immediate-Release Dosage Forms under Apparatus 4.

Delayed-Release Dosage Forms

Proceed as directed for Delayed-Release Dosage Forms under Apparatus 1 and Apparatus 2, using the specified media.

Time —Proceed as directed for Delayed-Release Dosage Forms under Apparatus 1 and Apparatus 2.

INTERPRETATION

Immediate-Release Dosage Forms

Unless otherwise specified in the individual monograph, the requirements are met if the quantities of active ingredient dissolved from the dosage units tested conform to Acceptance Table 1. Continue testing through the three stages unless the results conform at either S1 or S2. The quantity, Q, is the amount of dissolved active ingredient specified in the individual monograph,expressed as a percentage of the labeled content of the dosage unit; the 5%, 15%, and 25% values in Acceptance Table 1 are percentages of the labeled content so that these values and Q are in the same terms.

Acceptance Table 1

Stage	Number Tested	Acceptance Criteria
S1	6	Each unit is not less than Q + 5%.
S2	6	Average of 12 units (S1 + S2) is equal to or greater than Q, and no unit is less than Q 15%.
S3	12	Average of 24 units (S1 + S2 + S3) is equal to or greater than Q, not more than 2 units are less than Q 15%, and no unit is less than Q 25%.

Extended-Release Dosage Forms

Unless otherwise specified in the individual monograph, the requirements are met if the quantities of active ingredient dissolved from the dosage units tested conform to Acceptance Table 2. Continue testing through the three levels unless the results conform at either L1 or L2. Limits on the amounts of active ingredient dissolved are expressed in terms of the percentage of labeled content. The limits embrace each value of Qi, the amount dissolved at each specified fractional dosing interval. Where more than one range is specified in the individual monograph, the acceptance criteria apply individually to each range.

Acceptance Table 2

Level	Number Tested	Criteria
L1	6	No individual value lies outside each of the stated ranges and no individual value is less than the stated amount at the final test time.
L2	6	The average value of the 12 units (L1 + L2) lies within each of the stated ranges and is not less than the stated amount at the final test time; none is more than 10% of labeled content outside each of the stated ranges; and none is more than 10% of labeled content below the stated amount at the final test time.
L3	12	The average value of the 24 units (L1 + L2 + L3) lies within each of the stated ranges, and is not less than the stated amount at the final test time; not more than 2 of the 24 units are more than 10% of labeled content outside each of the stated ranges; not more than 2 of the 24 units are more than 10% of labeled content below the stated amount at the final test time; and none of the units is more than 20% of labeled content outside each of the stated ranges or more than 20% of labeled content below the stated amount at the final test time.

Delayed-Release Dosage Forms

NOT ACCEPTED BY THE JAPANESE PHARMACOPOEIA.

Acid Stage— Unless otherwise specified in the individual monograph, the requirements of this portion of the test are met if the quantities, based on the percentage of the labeled content, of active ingredient dissolved from the units tested conform to Acceptance Table 3. Continue testing through all levels unless the results of both acid and buffer stages conform at an earlier level.

Acceptance Table 3

Level	Number Tested	Criteria
A1	6	No individual value exceeds 10% dissolved.
A2	6	Average of the 12 units (A1 + A2) is not more than 10% dissolved, and no individual unit is greater than 25% dissolved.
A3	12	Average of the 24 units (A1 + A2 + A3) is not more than 10% dissolved, and no individual unit is greater than 25% dissolved.

Buffer Stage— Unless otherwise specified in the individual monograph, the requirements are met if the quantities of active ingredient dissolved from the units tested conform to Acceptance Table 4. Continue testing through the three levels unless the results of both stages conform at an earlier level. The value of Q in Acceptance Table 4 is 75% dissolved unless otherwise specified in the individual monograph. The quantity, Q,specified in the individual monograph is the total amount of active ingredient dissolved in both the Acid and Buffer Stages, expressed as a percentage of the labeled content. The 5%, 15%, and 25% values in Acceptance Table 4 are percentages of the labeled content so that these values and Q are in the same terms.

Acceptance Table 4

Level	Number Tested	Criteria
B1	6	Each unit is not less than Q + 5%.
B2	6	Average of 12 units (B1 + B2) is equal to or greater than Q, and no unit is less than Q – 15%.
B3	12	Average of 24 units (B1 + B2 + B3) is equal to or greater than Q, not more than 2 units are less than Q – 15%, and no unit is less than Q – 25%.

(Official April 1, 2006)

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1 The materials should not sorb, react, or interfere with the specimen being tested.

2 If a cover is used, it provides sufficient openings to allow ready insertion of the thermometer and withdrawal of specimens.

3 Test specimens are filtered immediately upon sampling unless filtration is demonstrated to be unnecessary. Use an inert filter that does not cause adsorption of the active ingredient or contain extractable substances that would interfere with the analysis.

4 One method of deaeration is as follows: Heat the medium, while stirring gently, to about 41, immediately filter under vacuum using a filter having a porosity of 0.45 µm or less, with vigorous stirring, and continue stirring under vacuum for about 5 minutes. Other validated deaeration techniques for removal of dissolved gases may be used.

727 CAPILLARY ELECTROPHORESIS

Electrophoresis refers to the migration of charged electrical species when dissolved or suspended in an electrolyte through which an electric current is passed. Cations migrate toward the negatively charged electrode (cathode), while anions are attracted toward the positively charged electrode (anode). Neutral particles are not attracted toward either electrode.

The use of capillaries as a migration channel in electrophoresis has enabled analysts to perform electrophoretic separations on an instrumental level comparable to that of high-performance liquid chromatography (HPLC), albeit with some distinct operational differences, advantages, and disadvantages relative to HPLC. This method of analysis is commonly known as capillary electrophoresis (CE). During typical CE operation with an uncoated capillary filled with a buffer, referred to as the “operating buffer,” silanol groups present on the inner wall of the glass capillary release hydrogen ions to the buffer and the wall surface becomes negatively charged, even at a fairly low pH. Cations, or solutes having partial positive charges in the medium, are electrostatically attracted to the negatively charged wall, forming an electrical double layer. The initiation of electrophoresis by applying voltage across the length of the capillary causes the solution portion of the electrical double layer to move toward the cathode end of the capillary, drawing the bulk solution. This movement of the bulk solution under the force of the electrical field is called the electroosmotic flow (EOF). The degree of ionization of the inner-wall capillary silanol groups depends mainly on the pH of the operating buffer and on the modifiers that may have been added to the electrolyte. At low pH, the silanol groups generally have a low ionization and the EOF is low. At higher pH, silanol groups become more ionized and the EOF increases. In some cases organic solvents, such as methanol or acetonitrile, are added to the aqueous buffer to increase the solubility of the solute and other additives or to affect the degree of ionization of the sample. The addition of such organic modifiers generally causes a decrease in the EOF. The detector is located toward the cathode end of the capillary. The EOF is usually greater than the electrophoretic mobility; thus, even anions are swept toward the cathode and the detector. When an uncoated capillary containing pH 7.0 phosphate buffer is used, the usual order of appearance of solutes in an electropherogram is cationic species, neutral solutes, and anionic species.

Currently, there are five major modes of operation of CE: capillary zone electrophoresis (CZE), also referred to as free solution or free flow capillary electrophoresis; micellar electrokinetic chromatography (MEKC); capillary gel electrophoresis (CGE); capillary isoelectric focusing (CIEF); and capillary isotachophoresis (CITP).

In CZE, separations are controlled by differences in the relative electrophoretic mobilities of the individual components in the sample or test solution. The mobility differences are functions of analyte charge and size under specific method conditions. They are optimized by appropriate control of the composition of the buffer, its pH, and its ionic strength.

In MEKC, ionic surfactants are added to the operating buffer at a concentration above their critical micelle concentration. The micelles provide a pseudostationary phase with which analytes can partition. This technique is useful for the separation of neutral and ionic species.

CGE, which is analogous to gel filtration, uses gel-filled capillaries to separate molecules on the basis of relative differences in their respective molecular weight or molecular size. It was first used for the separation of proteins, peptides, and oligomers. Gels may have the advantage of decreasing the EOF and also significantly reducing protein adsorption onto the inner wall of the capillary, which can significantly reduce analyte peak tailing effects.

In CIEF, substances are separated on the basis of their relative differences in isoelectric points. This is accomplished by achieving steady-state sample zones within a buffer pH gradient, where the pH is low at the anode and high at the cathode. The gradient is established by applying a voltage across a capillary filled with a mixture of carrier components consisting of amphoteric substances having different pI values.

CITP employs two buffers that enclose the analyte zones between them. Either anions or cations can be analyzed in sharply separated zones. In addition, the analyte concentrations are the same in each zone; thus, the length of each zone is proportional to the amount of the particular analyte.

The most commonly utilized capillary electrophoresis techniques are CZE and MEKC. These are briefly discussed in the following sections. Pertinent general principles and theory, instrumental considerations, analysis, and operational considerations and parameters are discussed as well.

PRINCIPLES OF CAPILLARY ZONE ELECTROPHORESIS

CZE makes use of the principles of electrophoresis and electroosmosis to achieve separation of charged species.

(1) The electrophoretic mobility of an ion, µEP, is described by the equation:

µEP = q / (6r),

in which q is the charge of the ion, is the solution viscosity, and r is the radius of the hydrated ion. This relationship infers that small, highly charged analytes have high mobilities and large, slightly charged analytes have low mobilities.

(2) The velocity of migration, EP, in cm per second, is represented by the equation:

EP = µEP(V / L),

in which µEP is the electrophoretic mobility; V is the applied voltage; and L, in cm, is the total capillary length.

(3) The velocity of the EOF, EO, in cm per second, is described by the equation:

EO = µEO(V / L),

in which µEO is the EOF mobility (the coefficient of electroosmotic flow), and the other terms are as defined above.

(4) The time, t, in seconds, necessary for a solute to migrate the entire effective length of the capillary (from the inlet to the detector), l, is represented by the relationship:

t = l / E(µEP + µEO) = lL / V(µEP + µEO),

in which E is the strength of the applied electrical field, and the other terms are as defined above.

(5) Efficiency of an electrophoretic system can be related to mobility and EOF and expressed in terms of the number of theoretical plates, N, by the equation:

N = (µEP + µEO)V / 2D,

in which D is the diffusion coefficient of the solute, and the other terms are as defined above.

(6) The resolution, R, of two consecutively eluting solutes can be defined by the equation:

where µEP1 and µEP2 are the mobilities of the two solutes,

is their average, and the other terms are as defined above.

PRINCIPLES OF MICELLAR ELECTROKINETIC CHROMATOGRAPHY

In MEKC, the supporting electrolyte medium contains a surfactant at a concentration above its critical micelle concentration (CMC). In this aqueous medium, the surfactant self-aggregates and forms micelles whose hydrophilic head groups form an outer shell and whose hydrophobic tail groups form a nonpolar core into which the solutes can partition. Generally, the micelles are anionic on their surface, and, under the applied voltage, they migrate in the opposite direction to the EOF. This type of partitioning is analogous to that in solvent extraction or reverse-phase HPLC. The differential partitioning of neutral molecules between the buffered aqueous mobile phase and the micellar pseudostationary phase is the sole basis for separation. The buffer and micelles form a two-phase system, and the analyte can partition between these two phases.

A micellar system suitable for MEKC meets the following criteria: the surfactant is highly soluble in the buffer, and the micellar solution is homogeneous and transparent when UV detection is employed. The most common surfactant for MEKC is sodium dodecyl sulfate (anionic surfactant). Others include cetyltrimethylammonium bromide (cationic surfactant) and bile salts (chiral surfactant). The selectivity of an MEKC system is mainly dependent on the nature of the surfactant. Organic solvents are often added to the MEKC buffer to adjust the capacity factors, just as in reverse-phase HPLC separations. MEKC may be used for the separation of enantiomers. For such separations, a chiral additive is added to the buffer or a chiral surfactant, such as a bile salt, is used.

A general knowledge of conventional column chromatographic principles aids in understanding MEKC principles. However, in MEKC the micelles are not truly stationary; therefore, the column chromatographic theory needs to be modified. The major modification introduced to MEKC principles is the finite nature of the separation window for neutral molecules.

(7) The migration time, tR, for a neutral species is expressed with the following equation:

tR = (1 + k¢)t0 / [1 + (t0 / tMC)],

in which t0 is the time required for an unretained substance to travel the effective length of the capillary; tMC is the time required for a micelle to traverse the capillary; k¢ is the capacity factor; and tR is always between t0 and tMC.

(8) The capacity factor, k¢, for a neutral species, is calculated by the equation:

k¢ = (tR / t0 1) / (1 tR / tMC),

in which the terms are as defined above.

(9) For practical purposes, k¢ is calculated by the equation:

k¢ = tR / t0 1,

in which tR is the time measured from the point of voltage application (or injection) to the peak maximum; and t0 is measured from the point of voltage application (or injection) to the leading edge of the solvent front or of an unretained substance. In contrast with CZE, k¢ in MEKC is significant and is a characteristic of a given solute in a given MEKC system. Further discussion of k¢ appears later in the System Suitability section under Operational Parameters.

(10) The resolution, RS, for neutral species is calculated by the equation:

in which is the selectivity, defined as the ratio of k¢2 to k¢1, of the operating conditions for separating two solutes. If the two solutes elute close together ( 1.1), either k¢ may be used. The equation shows that, just as with conventional chromatography, resolution in MEKC can be improved through controlling efficiency, selectivity, retention, and the chemical nature of the resolving surfactant-medium system. The last term of the equation is due to the limited elution range. Although MEKC is particularly useful in the separation of neutral species, this technique may also be used for the separation of charged solutes. The latter procedure involves a combination of chromatographic and electrophoretic separation mechanisms. The additional interaction between charged solutes and micelle can be used to optimize a separation. Ion-pairs may form if the charges borne on the surfactant and solute are opposite; otherwise, surfactant and solute repel each other. These differences can significantly influence the separation of charged molecules.

INSTRUMENTAL CONSIDERATIONS

A typical CE system (see Figure 1) contains a fused-silica capillary having an internal diameter of 50 to 100 µm and a length of 20 to 100 cm. The ends of the capillary are placed in separate electrolyte reservoirs. The direct-current power supply is capable of furnishing high voltages, typically ranging from 0 to 30 kV. A detector and autosampler with some form of data-recording device complete the system. An automatic buffer replenishment system and a computer-based control and data acquisition system may also be found on the standard commercial systems. Temperature controls for both the capillary and the autosampler are also available on commercial instruments.

The primary considerations of instrumentation include capillary type and configuration, modes of sampling, power supply and detector modes.

Fig. 1. Typical CE Instrument Configuration.

Capillary Type and Configuration

Capillaries used in CZE are usually made of fused silica and with no internal coating. Some instruments are configured with a “free-swinging” style of capillary; that is, the capillary is not encased within an enclosure. In most commercial instruments, the capillary is housed in a cartridge. Both configurations offer specific advantages and disadvantages. The ability of the instrument to accommodate different types of capillaries and capillaries of various diameters and lengths is an important consideration. Capillaries with a variety of internal coatings are also available; therefore, the ability of the instrument to accommodate different capillaries is important. Internal capillary coatings may be employed to alter the magnitude or direction of EOF or to reduce sample absorption. If an internally coated capillary is to be used, then sufficient details and the indication of the supplier must be included in the method. Capillaries from an alternate supplier can be used if it is demonstrated that they are suitable.

Sample Introduction and Injector Technology

Modes of sample introduction onto the capillary include electromigration (electrokinetic mode) and negative- and positive-pressure injection (hydrostatic mode).

For injection via electromigration, the sample solution is electrophoresed into the capillary by inserting the capillary and electrode into the sample vials and applying a brief, high voltage. The sample enters the capillary by a combination of electrophoresis and EOF. Therefore, analytes with different mobilities are loaded into the capillary to different extents. The conductivities of the sample and standard solutes also affect the EOF and the volume injected.

Negative-pressure injectors place negative pressure at the detector end of the capillary and draw the sample solution into the injection end of the capillary. Positive-pressure injectors pressurize the sample vial, forcing the sample into the capillary. Pressure injection loads all sample components into the capillary to the same extent, and it is generally the most reproducible and the most frequently applied injection mode. The sample volume injected depends on the capillary length and internal diameter and the voltage or pressure applied. The typical sample volumes injected into the capillary are between 1 and 20 nL.

Each injection method offers specific advantages and disadvantages, depending on the sample composition, the separation mode, and the application of the method. None of the above injection modes is as reproducible as commercially available HPLC injectors. Based on the circumstances, it may be necessary to use internal standards for specific methods where high injection precision is required.

Power Supply

Most commercially available CE units have direct-current power supplies that are capable of furnishing power on a ramp-up or step-function mode to achieve and maintain the desired operational voltage in a smooth manner. This will help to ensure a relatively smooth baseline.

Another essential feature of the power supply is its utility in introducing a sample at the cathodic or the anodic end of the capillary. Because it is impractical to relocate the on-line detector from one end of the instrument to the other, it is beneficial to be able to specify whether the sample injection end is at the cathode or the anode.

Detector Modes

CE systems generally offer UV-visible absorbance and laser-induced fluorescence (LIF) detectors. Scanning UV detectors or photodiode-array detectors are also available for many commercial CE instruments.

The coupling of CE to a mass spectrometer offers the possibility of obtaining structural information in conjunction with electrophoretic migration data.

Fluorescence detection offers an enhanced sensitivity for samples containing only very small amounts of UV-active analytes. Application of fluorescent tags to non-UV-absorbing compounds can be useful. Alternately, non-UV-absorbing or nonfluorescent analytes can be detected indirectly by adding a chromophore or a fluorophore, respectively, to the buffer: the non-absorbing species are detected through the absence of expected signal from the absorbing species. Conductivity and pulsed amperometric detectors can also be used but are not generally available on commercial CE instruments.

ANALYTICAL CONSIDERATIONS

Several parameters, namely, capillary dimensions, voltage, ionic strength, and pH, are optimized to give adequate resolution and separation. Care should be taken to avoid changes in temperature that will affect the viscosity of the buffer and, in turn, influence both the EOF and the solute mobilities.

Capillary Dimensions— Variation of the capillary diameter and length can affect the electrophoretic resolution. Increasing the capillary length results in longer migration times, usually increasing resolution and generating a lower current. Increasing the capillary diameter will usually increase current and associated internal temperature gradients that decrease resolution. Conversely, a reduction in capillary diameter will result in lower heat and better resolution. However, larger capillary diameters have advantages of better mass loading and improved signal-to-noise ratio.

Voltage Effects— When higher voltages are applied, additional internal heating of the operating buffer occurs because of the current flow through the buffer. This heating effect, known as Joule heating, must be controlled because resistance, dielectric constant, and viscosity are temperature-dependent and alter the velocity of the EOF and solute mobilities.

In general, increasing the voltage will result in increased efficiency and resolution (up to the point where Joule heat cannot be adequately dissipated). Maximum resolution is obtained by maintaining the voltage below the level at which Joule heating and diffusion become limiting factors.

Ionic Strength Effects— Control of ionic strength and its manipulation allow adjustment of resolution, efficiency, and sensitivity. Increasing ionic strength will generally improve resolution, peak efficiency, and peak shape. Sensitivity may be improved because better focusing is achieved. However, because the current generated is directly proportional to the buffer concentration, more heat is produced when ionic strength of the buffer is increased, hence limiting the ionic strengths that can be utilized.

pH Effects— Resolution, selectivity, and peak shape can be dramatically altered by changes in pH as this parameter affects the extent of solute ionization and the level of EOF. The EOF is high at high pH and low at low pH in uncoated fused-silica capillaries.

OPERATIONAL PARAMETERS

The major steps in operating a CE system are system setup, capillary rinsing procedure, running a sample, system suitability testing, sample analysis, data handling, and system shutdown.

System Setup— An appropriate capillary of specific length, inner diameter, and coating is selected, with considerations made for separation and resolution, ionic strength of buffer, and pH effects. A buffer of appropriate composition, ionic strength, and pH is prepared, degassed, if necessary, and passed through an appropriate filter. All solvents, including water, are HPLC or CE grade.

Capillary Rinsing Procedure— Improved consistency of migration times and resolution may generally be obtained if a defined rinsing procedure is followed. Capillary conditioning and rinsing procedures are very specific to the analyte, matrix, and method. Therefore, these procedures are developed as part of the method and are specified in the individual monograph. Rinsing may involve the use of solutions such as 0.1 M phosphoric acid, water, and 0.1 M sodium hydroxide. Before beginning analysis of the test specimen, the capillary may be rinsed with five column volumes of the operating buffer that is to be used for the test. When changing buffer composition, it is advisable to rinse the capillary with five column volumes of each new buffer to allow the capillary to be cleansed of the previous buffer. Use of a new uncoated fused-silica capillary usually requires a regeneration procedure to activate the surface silanol groups. This procedure may include an extended rinse with a sodium hydroxide solution. Coated capillaries are rinsed according to the manufacturer's guidelines because inappropriate rinsing can remove or damage the coating. Columns may be dedicated to particular methods or buffer types to prevent cross-contamination.

Running a Sample— An appropriate capillary, electrolyte, and injection procedure are selected to achieve adequate resolution, sensitivity, and separation, with well-shaped and well-defined peaks. The required injection precision for a specific method may require use of an internal standard. The internal standard is selected with consideration of its ability to adequately separate from the analyte. The performance of the system may be improved by rinsing the capillary between injections and supplying fresh buffer to the source and destination vials used during voltage application, namely, vials 2 and 4 in Figure 1. Replicate injections from the same sample vial may be performed provided that no cross-contamination occurs. If cross-contamination occurs, the capillary tip may be rinsed by briefly inserting it into a vial containing the buffer prior to inserting the capillary into the electrolyte or sample vial.

The operational parameters are specified in each individual monograph so as to minimize voltage effects, ionic strength effects, and pH effects. The instrument is set up to run with the appropriate capillary configuration and injection conditions, within the established linear dynamic range of the detector; and acceptable migration precision is ensured by appropriate choice of sample diluent, separation electrolyte, electrolyte additives, and capillary pretreatment conditions. Exercise caution to avoid overloading the capillary with sample, as this decreases efficiency and reproducibility.

System Suitability— Parameters measured may include injector reproducibility, system selectivity, system efficiency, and tailing. Resolution between the analytes and other compounds may be determined by using test mixture standards.

Parameters typically used to determine system suitability include relative standard deviation (RSD), capacity factor (k¢), the number of theoretical plates (N), sensitivity (limit of detection or quantitation), number of theoretical plates per meter (TPM), tailing factor (T), and resolution (R).

The peak shape is closely examined; ideally, the peak is symmetrical, with no shoulders and no excessive tailing. If these conditions are not met, corrective actions are taken before proceeding with the analysis. Peak integration is also closely examined to ensure that the peak response is correctly quantitated.

Replicate injections of a Standard preparation of known concentration can be used to determine the reproducibility of the CE system. Data from five or more replicate injections are used to calculate RSD. Unless otherwise specified in the individual monograph, the relative standard deviation for replicate injections is not more than 3.0%. Minimum injection precision values may be specified in specific CE methods, especially when determining trace-level components. Calculation of electrophoretic parameters in MEKC, as in other forms of CE, may involve a combination of chromatographic and electrophoretic relationships. Hence, capacity factor, k¢, for neutral analyte migration in MEKC can be calculated by the equation:

k¢ = tR – t0(1 – tR / tMC),

in which tR, t0, and tMC are the migration times of the analyte, the bulk solution (EOF), and the micelle, respectively.

The number of theoretical plates, N, is a measure of the efficiency of the system and is calculated by the equation:

N = 16(tR / W)2 or N = 5.54(tR / W1/ 2)2,

in which W is the analyte peak width at baseline, W1/2 is the analyte peak width at half-height, and tR is the analyte migration time.

The number of theoretical plates per meter, TPM, is a measure of the efficiency of the capillary as a function of peak width at baseline and can be calculated by the equation:

TPM = 1600(tR / W)2 / L,

in which L, in cm, is the total capillary length; and the other terms are as defined above. The tailing factor, T, of the analyte peak is a measure of peak symmetry, and it represents the degree of deviation of the symmetry of the peak from an ideally symmetrical Gaussian peak. This factor can be calculated by the equation:

T = W0.05 / 2f,

in which W0.05 is the length of a line constructed parallel to the peak base from the leading edge to the tailing edge of the peak at 5% of peak height; and f is the distance along the same line from the leading edge of the peak, appearing to the left of the peak maximum in the electropherogram, to the intercept of a perpendicular line dropped from the peak maximum to the base. A ratio of 1.0 indicates a perfectly symmetrical peak. If electrodispersive effects occur, they can generate highly asymmetrical peaks. This can occur when high sample concentrations are used, such as those for testing of impurities. Use of highly asymmetrical peaks is acceptable provided that they are reproducible and that they do not compromise separation selectivity.

The resolution factor, R, is a measure of the ability of the capillary system to separate consecutively migrating analytes. Resolution is determined for all sample analytes of interest, with the pH of the buffer adjusted as necessary to meet system suitability requirements. It can be calculated by the equation:

R = 2(t2 t1) / (W1 + W2),

in which t2 and t1 are the migration times, measured at peak maxima, for the slower migrating peak and the faster migrating peak, respectively; and W2 and W1 are the corresponding widths of these two peaks measured at their bases.

Sample Analysis— Once the suitability of the CE system has been established, aliquots of both the Standard preparation and the test preparation are injected. Standards are injected before or after the samples and intermittently throughout the run.

Data Handling— Time-normalized peak areas are often used in quantitative calculations. These are determined by dividing the observed integrated peak area by the migration time of the analyte. This compensates for the fact that in CE, unlike HPLC, each analyte travels through the detector at a different velocity. Unless this normalization is performed, slowly moving (later-migrating) analytes will have disproportionately large peak areas compared with those for early migrating components.

System Shutdown— After analysis, the capillary is rinsed according to the directions specified in each monograph or as recommended by the manufacturer. For example, the capillary might be rinsed with distilled water to remove buffer components and then filled with air or nitrogen by performing a rinse from an empty vial. Naturally, the destination and source vials, namely, vials 4 and 2 in Figure 1, are emptied of buffer and rinsed thoroughly with deionized water.

Auxiliary Information— Staff Liaison : Kahkashan Zaidi, Ph.D., Senior Scientific Associate

Expert Committee : (GC05) General Chapters 05

USP29–NF24 Page 2696

Phone Number : 1-301-816-8269