This article was downloaded by: [Shabir, Ghulam A.] On: 14 December 2009 Access details: Access Details: [subscription number 917720727] Publisher Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 3741 Mortimer Street, London W1T 3JH, UK Journal of Liquid Chromatography & Related Technologies Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713597273 A NEW VALIDATED METHOD FOR THE SIMULTANEOUS DETERMINATION OF A SERIES OF EIGHT BARBITURATES BY RPHPLC Ghulam A. Shabir a; Tony K. Bradshaw a; Shafique A. Arain b; Ghulam Qadir Shar c School of Life Sciences, Oxford Brookes University, Oxford, UK b Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, UK c Sunderland Pharmacy School, University of Sunderland, Sunderland, UK a Online publication date: 11 December 2009 To cite this Article Shabir, Ghulam A., Bradshaw, Tony K., Arain, Shafique A. and Shar, Ghulam Qadir(2010) 'A NEW VALIDATED METHOD FOR THE SIMULTANEOUS DETERMINATION OF A SERIES OF EIGHT BARBITURATES BY RP-HPLC', Journal of Liquid Chromatography & Related Technologies, 33: 1, 61 — 71 To link to this Article: DOI: 10.1080/10826070903430175 URL: http://dx.doi.org/10.1080/10826070903430175 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. 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The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Journal of Liquid Chromatography & Related Technologies, 33:61–71, 2010 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6076 print/1520-572X online DOI: 10.1080/10826070903430175 Downloaded By: [Shabir, Ghulam A.] At: 17:48 14 December 2009 A NEW VALIDATED METHOD FOR THE SIMULTANEOUS DETERMINATION OF A SERIES OF EIGHT BARBITURATES BY RP-HPLC Ghulam A. Shabir,1 Tony K. Bradshaw,1 Shafique A. Arain,2 and Ghulam Qadir Shar3 1 School of Life Sciences, Oxford Brookes University, Oxford, UK 2 Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, UK 3 Sunderland Pharmacy School, University of Sunderland, Sunderland, UK & A new reversed-phase high performance liquid chromatographic (RP-HPLC) method is developed and validated for the simultaneous determination of barbitone, allobarbitone, phenobarbitone, cyclobarbitone, hexobarbitone, pentobarbitone, secobarbitone and methohexitone compounds in a single analytical run. The method uses a Phenosphere C18 (150 mm  4.6 mm; 5 lm) column and isocratic elution. The mobile phase consisted of a mixture of methanol-water (50:50, v=v), pumped at a flow rate of 1.0 mL=min. The UV detection is set at 254 nm. The method is validated with respect to accuracy, precision (repeatability and intermediate precision), specificity, linearity, range robustness and stability of analytical solutions. All the parameters examined met the current recommendations for bioanalytical method validation. The method is specific, simple, selective and reliable for routine use in quality control analysis of barbiturates raw materials for final product release. Keywords allobarbitone, barbitone, cyclobarbitone, hexobarbitone, method development, method validation, methohexitone, pentobarbitone, phenobarbitone, reversedphase liquid chromatography, secobarbitone INTRODUCTION Barbiturates are widely in use since the beginning of the century (barbital, 1903) especially as sedative hypnotics. With the advent of anxiolytic agents the popularity of barbiturates has suffered although they are still less costly. However, those with specialized properties such as the anticonvulsant phenobarbital continue to be commonly used.[1] In addition, abuse of barbiturates is now widespread. Due to the international nature of the illegal drug market forensic laboratories encounter Correspondence: Ghulam A. Shabir, School of Life Sciences, Oxford Brookes University, Oxford OX3 0BP, UK. E-mail: gshabir@brookes.ac.uk 62 G. A. Shabir et al. Downloaded By: [Shabir, Ghulam A.] At: 17:48 14 December 2009 a vast range of such compounds. Complications arise from the fact that abused barbiturates often occur as complex mixtures and other drugs and=or excipients are also present.[2] This necessitates the continued development of methods for their efficient separation and precise identification. In this work a group of eight barbiturates was selected as model mixtures (Figure 1). Barbitone (BR) is also known as 5,5-diethylbarbituric acid, colours crystals or white crystalline powder with melting point 188–192 C. It is seldom used in modern therapeutics. Allobabitone (AB) known as 5,5-diallybarbituric acid, a white crystalline powder, melting point about 173 C, dissociation constant at pka 7.8 at 25 C. Phenobarbitone (PhB) is also known as 5-ethyl-5-phenylbarbituric acid, white crystalline powder, and melting point 174–178 C, pka 7.4 at 25 C. Cyclobarbitone (CB) is known as 5-(cyclohex-1-enyl)-5-ethylbarbituric acid, melting point FIGURE 1 Chemical structures of the barbiturates used in this study. Downloaded By: [Shabir, Ghulam A.] At: 17:48 14 December 2009 A New Validated Method for the Simultaneous Determination 63 171–175 C, pka 7.6 at 20 C. Hexobarbitone (HB) is known as 5-(cyclohex-1enyl)-1,5-dimethyl barbituric acid, melting point 144–148 C, pka 8.2 at 20 C. Pentobarbitone (PB) is known as 5-(ethyl-5(1-methylbutyl) barbituric acid, melting point from 127–133 C. Secobarbitone (SB) is known as 5-allyl-5-(1-methylbutyl) barbituric acid, melting point about 100 C, very slightly soluble in water, freely soluble in ethanol and ether, also soluble in chloroform, pka 7.9 at 20 C. Methohexitone (MH) is known as /-()5-allyl-1-methyl-5-(methypent-2-ynyl) barbituric acid. A white to faintly yellowish-white crystalline powder with melting point 92–96 C. Some methods for the determination of some barbiturates have been reported, such as micellar liquid chromatography,[3,4] supercritical fluid chromatography,[5] gas chromatography-mass spectrometry (GC=MS),[6] thin-layer chromatography (TLC),[7,8] high-performance thin-layer chromatography (HPTLC),[9] Capillary Electrophoresis (CE),[10] high-performance liquid chromatography (HPLC),[11–14] and gas chromatography (GC),[15,16] but simultaneous determination of a series of eight barbiturate by reversedphase HPLC and method validation has not been reported. Furthermore, most of these procedures reported require labour sample pre-treatment and solvent extraction or solid phase extraction. These pre-treatment steps are time-consuming, increase the error sources and make the procedure more laborious. Analytical methods validation is an important regulatory requirement in pharmaceutical analysis. In recent years, the International Conferences on Harmonization (ICH) has introduced guidelines for analytical methods validation[17] in Japan Europe and United States. The most widely applied analytical performance characteristics are accuracy, specificity, linearity, range, precision (repeatability and intermediate precision), stability of analytical solutions and robustness. The purpose of this study was to develop and validate a rapid, accurate, simple and robust reversed-phase HPLC method for the simultaneous determination of a series of eight barbiturates (barbitone, allobarbitone, phenobarbitone, cyclobarbitone, hexobarbitone, pentobarbitone, secobarbitone and methohexitone) in a single chromatographic run which can be reliably used in routine quality control analysis for final drug substances release for medicinal formulations. EXPERIMENTAL Materials All chemicals and reagents were of the highest purity. Methanol (HPLCgrade) and barbitone, allobarbitone, phenobarbitone, cyclobarbitone, hexobarbitone, pentobarbitone, secobarbitone and methohexitone were 64 G. A. Shabir et al. purchased from Sigma (Gillingham, UK). Distilled water was de-ionised by using a Milli-Q system (Millipore, Bedford, MA). Downloaded By: [Shabir, Ghulam A.] At: 17:48 14 December 2009 HPLC Instrumentation and Conditions A Knauer (Berlin, Germany) HPLC system equipped with a module 1000 LC pump, LC 3950 autosampler, LC 2600 photodiode-array (PDA) detector and a vacuum degasser. The data were acquired via Knauer ClarityChrom workstation data acquisition software. All chromatographic experiments were performed in the isocratic mode. The mobile phase consisted of a mixture of methanol-water (50:50, v=v). The flow rate was set to 1.0 mL=min. The injection volume was 10 mL and the detection wavelength was set at 254 nm. The chromatographic separation was carried out on a 150 mm
4.6 mm, 5 mm C18 Phenosphere column obtained from Phenomenex (Macclesfield, UK). Sample Preparation An accurately weighted amount (0.08 g) of barbitone, (0.039 g) allobarbitone, (0.037 g) phenobarbitone, (0.07 g) cyclobarbitone, (0.048 g) hexobarbitone, (0.070 g) pentobarbitone, (0.076 g) secobarbitone and (0.07 g) methohexitone were placed in a 100 mL volumetric flask and dissolved in mobile phase (stock). A 5 mL aliquot of stock solution was diluted to 100 mL in a volumetric flask in mobile phase, yielding a final concentration of 400, 195, 185, 35, 24, 35, 38, and 35 mg=mL, respectively. RESULTS AND DISCUSSION Method Development The chromatographic separation of barbitone, allobarbitone, phenobarbitone, cyclobarbitone, hexobarbitone, pentobarbitone, secobarbitone and methohexitone was carried out in the isocratic mode using a mixture of methanol-water (50:50, v=v) as mobile phase. The column was equilibrated with the mobile phase flowing at 1.0 mL=min for about 30 min prior to injection. The column temperature was ambient. 10 mL of standard solutions was injected automatically into the column. Subsequently, the liquid chromatographic behaviours of barbiturates were monitored with a PDA UV detector at 254 nm. Additionally, preliminary system suitability, precision, linearity, robustness and stability of solutions studies performed during the development of the method showed that the 10 mL injection volume was reproducible and the peak response was significant at the Downloaded By: [Shabir, Ghulam A.] At: 17:48 14 December 2009 A New Validated Method for the Simultaneous Determination 65 FIGURE 2 HPLC chromatogram of eight barbiturates: (1) barbitone, RT ¼ 2.82, (2) allobarbitone, RT ¼ 4.11, (3) phenobarbitone, RT ¼ 4.65, (4) cyclobarbitone, RT ¼ 6.68, (5) hexobarbitone, RT ¼ 8.83, (6) pentobarbitone, RT ¼ 11.52, (7) secobarbitone, RT ¼ 15.58 and (8) methohexitone, RT ¼ 24.63. analytical concentration chosen. Chromatograms of the resulting solutions gave good separation and resolution (Figure 2). The analysis time for standards for all compounds was ca. 30 min. System Suitability Test System suitability test was developed for the routine application of the assay method. Prior to each analysis, the chromatographic system must satisfy suitability test requirements (resolution and repeatability). Peakto-peak resolution, between each peak measured on a reference solution must be above 2. System suitability test was performed to determine the accuracy and precision of the system from six replicate injections of a solution containing 30 mg barbiturates=mL. All peaks were well resolved and the precision of injections for all preservative peaks were acceptable. The percent relative standard deviation (R.S.D.) of the peaks area responses were measured, giving an average between 0.9% and 0.36% (n ¼ 6). The tailing factor (T), capacity factor (K0 ), theoretical plate number (N) and height equivalent to a theoretical plate (HETP) were also calculated. The results of system suitability in comparison with the required limits are shown in Table 1. The proposed method met these requirements within the accepted limits.[18,19] 66 G. A. Shabir et al. TABLE 1 System Suitability Test Recommended Limits and Results of Eight Barbiturates Results of Barbiturates Parameters Retention time (min) Injection repeatability (n ¼ 6) Resolution (Rs) Capacity factor (K0 ) Tailing factor (T) HETP Theoretical plate (N) Downloaded By: [Shabir, Ghulam A.] At: 17:48 14 December 2009  Recommended Limits 1 2 3 4 5 6 7 8 – 2.82 4.11 4.65 6.68 8.83 11.52 15.58 24.63 R.S.D.  1 (%, n  5) 0.09 0.14 0.11 0.25 0.16 0.19 0.16 0.36 Rs > 1.5 >2 2 – >2000 – 2.65 0.625 0.015 2235 2.56 2.87 0.750 0.009 2491 2.06 3.37 0.750 0.009 2705 4.06 5.27 1.000 0.005 2852 4.30 7.30 0.887 0.004 3467 5.38 8.12 18.10 9.83 13.64 22.14 0.875 0.875 1.000 0.003 0.003 0.003 5023 5639 6737 Height equivalent to a theoretical plate. Robustness For the determination of method robustness within a laboratory during method development a number of chromatographic parameters were evaluated, such as flow rate, column temperature, mobile phase composition, columns from different batches, and the quantitative influence of the variables were determined. For each parameter studied two injections of standard solutions were chromatographed. In all cases the influence of the parameters were found within a previously specified tolerance range. This shows that the method for determination of barbitone, allobarbitone, phenobarbitone, cyclobarbitone, hexobarbitone, pentobarbitone, secobarbitone and methohexitone was reproducible and robust. METHOD VALIDATION Linearity and Range The linearity test was performed using five different amounts of barbitone, allobarbitone, phenobarbitone, cyclobarbitone, hexobarbitone, in the range 360–440, 155–230, 155–220, 5–65, 10–40 mg=mL, respectively and for pentobarbitone, secobarbitone and methohexitone 5–65 mg=mL. Solutions corresponding to each concentration level were injected in duplicate and linear regression analysis of the barbiturates peak area (y) versus barbiturates concentration (x) was calculated. The correlation coefficients (r2 ¼ 0.9995) obtained for each barbiturate for the regression line demonstrates that there is a strong linear relationship between peak area and concentration of barbiturates (Table 2). 67 A New Validated Method for the Simultaneous Determination TABLE 2 Linearity Assessment of the HPLC Method for the Assay of Barbiturates Components Barbitone Allobarbitone Phenobarbitone Cyclobarbitone Hexobarbitone Pentobarbitone Secobarbitone Methohexitone Downloaded By: [Shabir, Ghulam A.] At: 17:48 14 December 2009 a Concentration (mg=mL)a Range (mg=mL) Equation for regression line r2 400 195 185 35 24 35 38 35 360–440 155–230 155–220 5–65 10–40 5–65 5–65 5–65 y ¼ 31.08x  10591 y ¼ 33.118x  4630.3 y ¼ 44.533x  6488.2 y ¼ 44.18x þ 63.3 y ¼ 80.876x  370.58 y ¼ 44.76x þ 76.8 y ¼ 44.087x þ 73.167 y ¼ 44.687x þ 57.967 0.9999 0.9996 0.9998 0.9995 0.9995 0.9997 0.9998 0.9997 Target concentration corresponding to 100%. Precision The precision of the method was determined by repeatability (intra-day) and intermediate precision (inter-day variation). Repeatability was examined by analysing six determinations of the same batch of each component at 100% of the test concentration. The relative standard deviation (R.S.D.) of the areas of barbiturates peak were found to be less than 0.36% (Table 3), which confirms that the method is sufficiently precise. Intermediate precision (inter-day variation) was studied by assaying five samples containing the nominal amount of barbiturates on different days. Solutions corresponding to each concentration level were injected in duplicate. The R.S.D. values across the system were calculated and found to be less than 0.45% (Table 3) for each of the multiple sample preparation, which demonstrates excellent precision for the method. TABLE 3 Method Validation Results for Barbiturates Results Validation Steps Repeatability Int. precision Day 1 Day 2 Standard stability (24 h data) System suitability Parameters 1 2 3 4 5 6 7 8 R.S.D. (%, n ¼ 6) 0.22 0.13 0.19 0.35 0.20 0.11 0.19 0.26 R.S.D. (%) R.S.D. (%) Change in response factor (%) R.S.D. (%, n ¼ 6) 0.22 0.27 0.10 0.18 0.22 0.09 0.24 0.18 0.13 0.17 0.13 0.15 0.09 0.14 0.11 0.11 0.39 0.14 0.22 0.27 0.12 0.19 0.45 0.13 0.18 0.18 0.23 0.16 0.18 0.32 0.22 0.29 68 G. A. Shabir et al. TABLE 4 Recovery Studies of the HPLC Method for the Assay of Barbiturates Applied Concentration (% of Target) (n ¼ 3) Components 50 (%) Barbitone Allobarbitone Phenobarbitone Cyclobarbitone Hexobarbitone Pentobarbitone Secobarbitone Methohexitone 99.98 99.97 99.93 99.92 99.82 99.98 99.84 99.95 (0.14)a (0.21) (0.16) (0.17) (0.37) (0.23) (0.09) (0.39) 100 (%) 99.92 100.00 99.92 100.00 99.92 98.94 99.97 100.00 (0.17) (0.32) (0.27) (0.32) (0.44) (0.30) (0.27) (0.21) 150 (%) 99.88 99.84 99.80 100.00 99.85 99.72 99.79 99.84 (0.27) (0.35) (0.17) (0.15) (0.17) (0.25) (0.48) (0.25) a Downloaded By: [Shabir, Ghulam A.] At: 17:48 14 December 2009 The coefficient of variation. Accuracy/Recovery Study Recovery studies may be performed in a variety of ways depending on the composition and properties of the sample matrix. In the present study, three different solutions were prepared with a known added amount of pure barbiturate compounds to give a concentration range of 50–150% of that in a test preparation. These solutions were injected in triplicate and percent recoveries of response factor (area=concentration) were calculated (Table 4). Specificity and Selectivity Injections of the blank were performed to demonstrate the absence of interference with the elution of the barbitone, allobarbitone, phenobarbitone, cyclobarbitone, hexobarbitone, pentobarbitone, secobarbitone and methohexitone barbiturates. These results demonstrate (Figure 3) that there was no interference from the other compounds and, therefore, confirm the specificity of the method. Stability of Analytical Solutions Sample solutions were chromatographed immediately after preparation and then re-assayed after storage at room temperature for 24 h. The results given in Table 3 showed there was no significant change (<0.16% response factor) in barbiturate concentrations over this period. Measurement of Robustness Analytical methods developed for use in quality control laboratories ideally are robust. Retention time for the analytes of interest will not Downloaded By: [Shabir, Ghulam A.] At: 17:48 14 December 2009 A New Validated Method for the Simultaneous Determination 69 FIGURE 3 HPLC chromatogram of the blank run. change significantly from day-to-day or from laboratory-to-laboratory if the method is considered robust. To determine the robustness of the chromatographic methodology developed for barbiturates, experimental conditions were purposely altered and chromatographic characteristics were evaluated. The effected temperature was also studied. Standard solutions were prepared and injected at early 20 C and again at 27 C. In all cases studied, the retention times of these compounds (barbitone, allobarbitone, phenobarbitone, cyclobarbitone, hexobarbitone, pentobarbitone, secobarbitone and methohexitone) were remains same 2.83, 4.12, 4.66, 6.68, 8.83, 11.51, 15.57 and 24.64 min, respectively (Figure 4). The coefficient of variation for retention time was lass then 1%. Good separation was always achieved, indicating that the analytical method remained selective for all components under the measured conditions. System Suitability A system suitability test was performed to determine the accuracy and precision of the system by injecting six replicate injections of barbitone, allobarbitone, phenobarbitone, cyclobarbitone, hexobarbitone, pentobarbitone, secobarbitone and methohexitone standard solutions. The R.S.D. of the peak areas responses was measured. The R.S.D. for barbiturates was less then (0.33%) as can be seen in Table 3. Downloaded By: [Shabir, Ghulam A.] At: 17:48 14 December 2009 70 G. A. Shabir et al. FIGURE 4 Retention times (min) of barbiturates: barbitone (BR), allobarbitone (AB), phenobarbitone (PhB), cyclobarbitone (CB), hexobarbitone (HB), pentobarbitone (PB), secobarbitone (SB), and methohexitone (MH). CONCLUSION A new RP-HPLC method with UV spectrophotometric detection was developed successfully for the simultaneous determination of a series of eight barbitone, allobarbitone, phenobarbitone, cyclobarbitone, hexobarbitone, pentobarbitone, secobarbitone and methohexitone compounds. The method was validated and the results obtained were accurate and precise with R.S.D. < 1% in all cases and no significant interfering peaks were detected. The method is specific, simple, selective and reliable for routine use in quality control analysis of barbiturates raw materials for final product release. REFERENCES 1. Gringauz, A. In Introduction to Medicinal Chemistry; Wiley-VCH: 1997; 568–569. 2. Gill, R.; Stead, A.H.; Moffat, A.C. Analytical aspects of barbiturate abuse: Identification of drugs by the effective combination of gas-liquid, high-performance liquid and thin-layer chromatographic techniques. J. Chromatogr. A. 1981, 204, 275–284. 3. Marina, D.; Rukhadze, G.; Bezarashvili, S.; Maya, V. S.; Veronika, R. M. Separation of barbiturates with micellar liquid chromatography and optimization by a second order mathematical design. J. Chromatogr. A. 1998, 805, 45–53. 4. Martı́n, S.; Sagrado, R. M.; Villanueva, C.; Medina, M.J. Determination of barbiturates in urine by micellar liquid chromatography and direct injection of sample. J. Pharm. Biomed. Anal. 1999, 21, 331–338. 5. Roger, M.; Smith, M.; Marsin, S. Application of packed column supercritical fluid chromatography to the analysis of barbiturates. J. Pharm. Biomed. Anal. 1988, 6, 837–841. Downloaded By: [Shabir, Ghulam A.] At: 17:48 14 December 2009 A New Validated Method for the Simultaneous Determination 71 6. Brad, J.H.; Jennifer, S. B. Determination of barbiturates by solid-phase microextraction (SPME) and ion trap gas chromatography–mass spectrometry. J. Chromatogra. A. 1997, 777, 275–282. 7. Tibor, C.; Jacek, B.; Éva, F.; Jozsef, S. Reversed-phase thin-layer chromatography of barbiturates in the presence of soluble b-cyclodextrin polymer. J. Chromatogra. A. 1986, 351, 356–362. 8. Grassini, G.S.; Cristalli, M. Comparison of Cn bonded silica gel thin-layer chromatographic plates: conditions for use and separations of some barbiturates. J. Chromatogra. A. 1981, 214, 209–216. 9. Giuliana, G.S.; Isabella, N. High-performance thin-layer chromatography on amino-bonded silica gel: application to barbiturates and steroids. J. Chromatogra. A. 1985, 322, 149–158. 10. Ting-Fu, J.; Yuan, H.W.; Zhi, L.; Mei, E.Y. Direct determination of barbiturates in urine by capillary electrophoresis using a capillary coated dynamically with polycationic polymers. Chromatographia. 2007, 65, 611–615. 11. Fernandez, G.B.P.; Lores, M.; Cela, R. Analysis of barbiturates by micro-high-performance liquid chromatography with post-column photochemical derivatization. J. Chromatogra. A. 2000, 870, 39–44. 12. Kyoko, I.; Yoko, K.; Hiroko, M.; Toshiko, U. Determination of barbiturates in mouse tissue by high-performance liquid chromatography. J. Chromatogra. A. 1981, 205, 401–412. 13. White, P.C. Use of dual-wavelength UV detection in high-performance liquid chromatography for the identification of barbiturates. J. Chromatogra. A. 1980, 200, 271–276. 14. Hulshoff, A.H.; Roseboom, J.R. Improved detectability of barbiturates in high-performance liquid chromatography by pre-column labelling and ultraviolet detection. J. Chromatogra. A. 1979, 186, 535–541. 15. Dilranjan, N.; Pillai, S.D. Analysis of barbiturates by gas chromatography. J. Chromatogra. A. 1981, 220, 253–274. 16. Mats, G.; Inga, P. Gas chromatographic determination of barbiturates by extractive alkylation and support coated open tubular column separation. J. Chromatogra. A. 1977, 140, 165–169. 17. International Conference on Harmonization (ICH), Q2 (R1): Validation of analytical procedures: Text and Methodology, November 2005. 18. Reviewer Guidance: Validation of Chromatographic Methods, Food and Drug Administration (FDA), Center for Drug Evaluation and Research (CDER), November 1994. 19. Shabir, G.A. Validation of high-performance liquid chromatography methods for pharmaceutical analysis: Understanding the differences and similarities between validation requirements of the US Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. J. Chromatogr. A. 2003, 987, 57–66.