5.1 Introduction to Spiro alcohol and a brief note on Trospium. Spiro alcohol, chemically described as 3a-hydroxynortropane-8-spiro-

Transcription

1 Introduction to Spiro alcohol and a brief note on Trospium chloride Spiro alcohol, chemically described as 3a-hydroxynortropane-8-spiro- 1 -pyrrolidinium chloride is a key intermediate of Trospium chloride. Trospium is a quaternary ammonium compound that has antispasmodic, antimuscarinic effects, but immediately crosses the blood brain barrier. Trospium chloride (INN) is a muscarinic antagonist urinary antispasmodic. It has been developed and patented by Robert Pfleger (Germany) and is sold under the brand name Sanctura in the US, Tropez OD in India, Trosec in Canada, Regurin and Flotros in the United Kingdom and Spasmex in Germany, Russia, Turkey, Argentina, Chile and Israel. The drug is used for the treatment of overactive bladder.it is available in Egypt under the brand name Trospikan by Hikma pharma. SANCTURA, a muscarinic antagonist, is indicated for the treatment of overactive bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency. The recommended dose is 20 mg twice daily.[1] An overactive bladder is a condition that results from sudden, involuntary contraction of the muscle in the wall of the urinary bladder. Overactive bladder causes a sudden and unstoppable need to urinate (urinary urgency), even though the bladder may only contain a small amount of urine.

2 178 The aim of the present study is to separate and quantify the impurities in spiro alcohol by a simple and novel technique coupled with estimation of spiro alcohol in trospium chloride. The molecular formula of Spiro alcohol is C11H20ClNO and molecular weight is Fig.5.1 Chemical structure of Spiro alcohol Structure and Chemical name Molecular weight/molecular formula MolecularFormula: C11H20ClNO Chemical name: (1R,3r,5S)-3-hydroxyl spiro[bicyclo[3.2.1]octane-8,1'- pyrrolidin]-1'-ium chloride Molecular Weight: A Versatile, Validated Method for Separation and Quantification of Spiro Alcohol and its Related Substances by HPLC-ELSD Materials and Reagents: Samples of Spiro alcohol and its three known impurities namely Impurity-1, Impurity-2 and Impurity-3 [Fig.1] were received from Process Research Department of Active Pharmaceutical ingredients of Dr. Reddy s Laboratories Limited, Hyderabad, India. HPLC grade Formic acid and Triethylamine were purchased from Merck, Schuchardt OHG, Germany.

3 179 HPLC grade Acetonitrile was purchased from Rankem, India. High pure water was prepared by using Millipore Milli Q plus purification system Equipment: The LC system, used for method development, and method validation was Agilent 1100 series (manufactured by Agilent Technologies, Waldron, Germany) LC equipped with a ELS detector model: 2000ES and model: 3300 (manufactured by Alltech). The output signal was monitored and processed using Empower software(waters) on Pentium computer (Digital Equipment Co) Chromatographic conditions: The chromatographic separation was achieved on an ACE C8 250mm x 4.6mm, 5µm column using a mobile phase containing a mixture of 0.1% v/v each of triethyl amine and formic acid in water as solvent-a and further using a mixture of solvent-a and acetonitrile in the ratio (70:30) (v/v) as solvent-b. The mobile phase was filtered through a nylon membrane (pore size 0.45µm) and degassed with helium spurge for 5min.The gradient programme : T (min) / % B: 0.01/2, 5/2, 8/50, 15/2 and 20/2 with flow rate of 0.6 ml/min was employed. The HPLC column was maintained at 25 C and the detector settings for Alltech 3300 model are Drift tube temperature: 52 C, Nitrogen gas flow: 1.5 L/min, Gain: 1. the injection volume was 5µL. Water was used as diluent during the standard and test samples preparation.

4 180 A brief synthetic scheme of Trospium chloride is shown in Fig Fig.5.2 A brief synthetic scheme of Trospium chloride

5 181 Fig.5.3 Chemical structures of impurities of Spiro alcohol Impurity name Impurity-1 (Nortropine) Structure and Chemical name HN OH Chemical name: (1R,3r,5S)-8- azabicyclo[3.2.1]octan-3-ol Molecular weight/molecular formula MolecularFormula: C7H13NO Molecular Weight: Impurity-2 (Tropine) Impurity-3 (Exo isomer) Chemical name: (1R,3r,5S)-8-methyl-8- azabicyclo[3.2.1]octan-3-ol Chemical name: (1R,3s,5S)-3-hydroxyspiro [bicyclo[3.2.1] octane-8,1'-pyrrolidin]-1'-ium chloride MolecularFormula: C8H15NO Molecular Weight: MolecularFormula: C11H20ClNO Molecular Weight:

6 Sample preparation: Standard and test solutions of 10mg/mL were prepared individually and a stock solution of impurity blend (mixture of Imp-1, Imp-2, Imp-3 and Spiro alcohol) 1000 µg/ml was prepared as well in diluent. Working solutions of impurity blend (mixture of Impurity-1, Impurity- 2, Impurity-3 and Spiro alcohol) at 2.5µg/mL, 5.0µg/mL, 7.5µg/mL, 10.0µg/mL, 12.5µg/mL and 15.0µg/mL were also prepared in diluent. Standard and test solutions of 1000 µg/ml were prepared individually and used for assay determination. 5.3 Method development approach and selection of suitable chromatographic technique: Preliminary HPLC experiments were performed by using RI detector with mobile phase of Triethyl amine and Formic acid buffer mixed with acetonitrile. All the experiments were relentlessly endured from the negative peaks near to the analyte peaks, which could lead to quantification errors during integration. More over, drawback for gradient mode also restricted to proceed further. Initial GC experiments discovered that a direct GC measurement with a flame ionization detector is not practical for the Spiro alcohol, because of extremely high non-volatility of this quaternary compound. Efforts were initiated to break the salt selectively to increase volatility by using quantitative amount of alkaline solution. These experiments endured from slight degradation of compound which gave inaccurate results. It

7 183 was established by analyzing spectrally pure compound (about 99% by NMR) having purity with 92% and two major peaks about 3 to 4%, which are not reflected in NMR results. This data confirmed the degradation of compound during the GC analysis. In addition, separation of Exo isomer is also a limitation, though by varying a range of stationary phases and temperature programs. Thus the trial prevent a consistent and simultaneous analysis of Spiro alcohol related substances and assay of Spiro alcohol. Due to this cause, the choice of GC-FID analysis for this compound has been proved futile. To enhance the sensitivity against UV, trials were made to the selective site pre column derivatization of OH group in the Spiro alcohol. Quantification of Spiro alcohol was achieved by derivatisation of analyte with benoxoprofen reagent was detailed in the literature[2]. In this method, benoxoprofen reagent reacts with OH group in analyte to get fluorescent nature. The procedure involved a number of pre column derivatisations and solvent extractions especially to increase the sensitivity and selectivity with poor UV or fluorescent characteristic of compound. In addition, the pre column derivatisation would limit the specificity on detection system and they do not authenticate complete conversion. Recently successful application of the HPLC coupled with evaporative light scattering detector (ELSD) would be expected to provide a universal

8 184 response to all analytes having lower volatility than the mobile pahse [3].The HPLC-ELSD also present several advantages in comparison to RI detector in terms of much higher sensitivity for solutes and high baseline stability even though it is operated under an isocratic or gradient elution mode [4].Because of the lack of chromophoric group, the use of an ELSD, therefore, appeared to be a favorable choice for their direct and high sensitive detection. In view of the fact that the nebulizing gas flow (N2) rate and evaporating temperature in an ELSD influence significantly in the rate of efficiency of response such as detection sensitivity and stability of base line, initial trail was kept to optimal conditions of the detector settings. To enhance the sensitivity for ELSD, the flow rate was held as low as possible at 1.5 L/min, the drift tube temperature at 70 C and gain 1 for Alltech 3300 model detector. The major objective is separation of Exo isomer, tropine and nortropine from Spiro alcohol. Different chromatographic conditions like different mobile phase compositions, different buffers, various ph and various diluents were evaluated. Trials were also made by using various stationary phases like C18, AQ, Phenyl and Cyano. The major problem found during method development is separation of Exo isomer and early elution of analyte peaks. Interestingly HCl also provided signals in ELSD, which were interfering with Exo compound. Finally separation was

9 185 achieved on C8 column with an isocratic elution of a mixture of Triethyl amine-formic acid (0.1% each in aqueous) and Methanol in the ratio of (95:5) with Alltech 3300 detector. In the ruggedness study, the same separation was evaluated in Alltech 2000ES detector by using same conditions. Interestingly, no signal appeared for nortropine in standard solution. It is due to the difference in drift tube lengths between two detector models. To a certain extent, less temperature is required to get the signal for nortropine in advanced detector. Hence drift tube temperature reduced to 52 C from 70 C, but simultaneously HCl peak response increased drastically and peak width increased and merged with Exo isomer. To achieve separation between Exo isomer and HCl, the chromatographic conditions are again optimized. Finally very good separation was achieved on a C8 column using a mobile phase containing a mixture of 0.1% v/v each of triethyl amine and formic acid in water as solvent-a and further using a mixture of solvent-a and acetonitrile in the ratio (70:30) (v/v) as solvent-b with gradient elution Optimised chromatographic conditions for the determination of spiro alocohol and its related impurities by ELSD: Column Flow rate Column temp : ACE C8 250mm*4.6mm*5.0µm : 0.6mL/min : 25±2 C

10 186 Injection volume : 5µL Diluent : Water Preparation of Solvent-A: A mixture of 0.1% v/v each of triethyl amine and formic acid in water Preparation of Solvent-B: A mixture of Solvent-A and acetonitrile in the ratio (70:30) (v/v) Table 5.1: Gradient programme Time (min) %Solvent-A %Solvent-B ELSD Settings for Alltech 3300 model: Drift tube temperature : 52 C Nitrogen gas flow : 1.5 L/min Gain : 1 Fig.5.4 Typical spiked and individual impurities chromatograms

11 187

12 Analytical method validation- Results and Discussion: The method that was developed and optimized in HPLC was considered for method validation. The analytical method validation was carried out in accordance with ICH guidelines.[5-6] System suitability test: System suitability testing is an integral part of chromatographic method. The tests are based to ensure that the equipment, analytical operations, electronics and samples to be analysed make an integral system and it can be calculated as such. The Spiro alcohol was spiked with 0.10% exo isomer with respect to the concentration of Spiro alcohol and injected for three times into HPLC system. Resolution between Spiro alcohol and exo isomer, tailing factor for Spiro alcohol was calculated. Good resolution was obtained between Spiro alcohol and exo isomer [Fig: 5.4]. System suitability results were tabulated [Table: 5.2].

13 189 Table 5.2: Results of system suitability Compound (n=3) Retention time (Rt) Resolution between Spiro alcohol and exo isomer (Rs) Tailing factor (T) Spiro alcohol Exo isomer Limit of Quantification (LOQ) and Limit of Detection (LOD): LOQ and LOD established for all impurities based on the impurities dilution linearity method Methodology for establishment of LOQ and LOD: The LOQ of an analytical method is the lowest concentration of analyte in a sample that can be quantitatively determined with appropriate precision and accuracy. The LOQ is a parameter of quantification used particularly for the determination of impurities. LOD and LOQ are determined by injecting linear solutions of all impurities known concentrations using ten levels ranging from 1.0µg/mL to 10.0µg/mL. The calculation method is based on the standard deviation (SD) of the response and the slope (S) of the calibration plot and using the formula LOQ=10xSD/S and LOD=3.3x SD/S.

14 190 Table 5.3: LOQ and LOD values of the impurities S.No. Impurity/product name LOQ in µg/ml LOD in µg/ml 1 Imp Imp Imp Spiro alcohol Precision at Limit of Quantification level: The precision of related substances method at LOQ level was determined by injecting six individual preparations of Imp-1, Imp-2 and Imp-3 with respect to the spiro alcohol concentration. Upon repetitive injections at quantification limit, the peak properties (retention time, area) were not influenced. Results have shown negligible variation in measured responses which revealed that the method was repeatable at LOQ level with RSD below 2.5%. The % RSD of area of Imp-1, Imp-2 and Imp-3 for six consecutive injections was 1.6, 2.5 and 1.3 respectively [Table 5.4]. Table 5.4: Precision results of all impurities at LOQ level S.No. Imp-1 Imp-2 Imp-3 Prep Prep Prep Prep Prep Prep Average Stdev %RSD

15 Accuracy at Limit of Quantification level: Standard addition and recovery experiments were performed to evaluate accuracy of the developed method for the quantification of all impurities in spiro alcohol sample at LOQ level. The recovery study for all impurities was carried out in triplicate at LOQ level of the target spiro alcohol concentration ( 10 mg/ml). The method showed constant and high absolute recovery at LOQ level with a mean absolute recovery of %. [Table 5.5] Table 5.5: Recovery at LOQ level for impurities 1-3 S.No. Impurity name % Recovery 1 Imp Imp Imp Precision: The precision of an analytical method convey the closeness of agreement (degree of scatter) between the series of measurements acquired from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be measured at three levels: repeatability, intermediate precision and reproducibility.it is normally expressed as RSD%. Repeatability is the results of a method operated over a short interval of time under the same conditions.

16 192 Intermediate precision is the end result from within-laboratories variations due to random events that include different days, different analysts, different equipment, etc. Reproducibility is determined by testing the homogeneous samples in different laboratories. It is a measure of precision between laboratories. The precision of the related substance method was evaluated by injecting six individual preparations of Spiro alcohol(10 mg ml -1 ) spiked with 0.10% of impurity-1, impurity-2 and impurity-3 with respect to Spiro alcohol analyte concentration. The % RSD for content of all impurities for six consecutive determinations was below 0.7[Table 5.6]. Table 5.6: Precision results of the RS method S.No. Imp-1 Imp-2 Imp Mean Stdev %RSD

17 193 Assay method precision study was evaluated initially by performing system precision, then by carrying out six independent assays of Spiro alcohol test sample against qualified reference standard and RSD of six consecutive assays was 0.1% [Table 5.7]. Results showed insignificant variation in measured response which demonstrated that the method was repeatable with RSD below 0.1%. Table 5.7: Precision results of the assay method Preparation Mean Stdev %RSD % Assay Intermediate precision for assay method was performed by carrying out six independent assays of Spiro alcohol test sample against qualified reference standard and calculated RSD of six consecutive assays. Related substances method was also performed by injecting six individual preparations of Spiro alcohol(10 mg/ml) and 0.10% of impurity-1, impurity-2 and impurity-3 with respect to Spiro alcohol analyte

18 194 concentration over different day, different instrument and with different analyst [Table 5.8]. Table 5.8: Results of Intermediate Precision Study S.No. Parameter Change 1 Different Analyst (a) Analyst-1 (a) Analyst-2 %RSD for Assay %RSD for Related Substances <0.7 <0.4 2 Differnet Instrument (a) Agilent 1100 series with ELSD model 3000 (b) Agilent 1100 series with ELSD model 2000ES <0.7 <0.6 3 Different Day (a) Day-1 (b) Day <0.7 < Linearity: Linearity of the related substances method: The linearity of an analytical method is the ability to attain test results which are directly proportional to the concentration of analyte with in the given range. Detector response linearity experiments were carried out by preparing the Spiro alcohol sample solution containing Imp-1, Imp-2 and Imp-3 covering the range from LOQ 150% (LOQ, 0.025, 0.05, 0.075, 0.10,

19 and 0.15%) with respect to specification limit (0.10%), was assessed by injecting seven separately prepared solutions of the normal test sample concentration (10 mg/ml). The correlation coefficients, slopes and Y-intercepts of the calibration curve were determined [Table ]. The Calibration curve was drawn by plotting average area of the impurity (Imp-1, Imp-2 and Imp-3) on the Y-axis and concentration on the X-axis [Fig. 5.5] which has shown linear relationship with a regression coefficient greater than for all impurities. Table 5.9: Linearity of Imp-1 %Conc. w.r.t Spiro Concentration in mg/ml Area alcohol LOQ correlation coefficient slope y-intercept % y-intercept at 100% level -0.56

20 196 Table 5.10: Linearity of Imp-2 %Conc. w.r.t Spiro Concentration in mg/ml Area alcohol LOQ correlation coefficient slope y-intercept % y-intercept at 100% level 0.40 Table 5.11: Linearity of Imp-3 %Conc. w.r.t Spiro Concentration in mg/ml Area alcohol LOQ correlation coefficient slope y-intercept -8.6 % y-intercept at 100% level -0.03

21 197 Fig 5.5: Linearity graph for Imp-1 to Imp Linearity of the assay method: The linearity of the assay method was established by injecting test sample at the level of 25%, 50%, 75%, 100%, 125% and 150 % of Spiro alcohol assay concentration (i.e 1000 µg/ml). Each solution was injected thrice (n=3) into LC system and the average area at each concentration each calculated. Calibration curve obtained by least square regression analysis between average peak areas and the concentration shown [Fig.

22 ]. A linear relationship with regression coefficient of greater than The best fit linear equation obtained was y = 2560x [Table 5.12] Table 5.12: Linearity results of the assay method Concentration (µg/ml) Average Peak area correlation coefficient slope y-intercept % y-intercept at 100% level Fig: 5.6: Linearity plot for Assay Method Accuracy: The accuracy of an analytical method is measure of the closeness of test results obtained to the true value.

23 Accuracy of the related substances method: The accuracy of the RS method calculated at 50%, 100% and 150% to the impurities specification limit (0.10%). The test solution prepared in triplicate (n=3) with impurities (Imp 1-3) at the level of 0.05%, 0.10% and 0.15% (w.r.t 10 mg/ml test concentration). The mean % recovery of impurities determined in the spiked test solution by using the area of impurities in the standard solutions at 0.10% level with respect to Spiro alcohol analyte concentration [Table 5.13]. Table 5.13: Recovery at 50%, 100% and 150 % level Impurity Name Imp-1 Imp-2 Imp-3 Spike level (%) Added (µg/ml) Recovered (µg/ml) Recovery (%) The related substances method have revealed consistent and high recoveries at all the three concentration levels i.e 50%, 100% and 150%, which convey the absolute recovery ranging from 98.5% to 102.4%. The

24 200 Spiro alcohol recovery study specified that the related substances by LC method were appropriate for determination/quantification of impurities in Spiro alcohol Accuracy of the assay method: Accuracy of the assay was performed by injecting three preparations of test sample at the level of 50%, 100% and 150% of analyte (Spiro alcohol test concentration) i.e 10 mg/ml. The study was performed in triplicate (n=3), the solution was injected into HPLC system and the mean peak area of Spiro alcohol analyte peak was calculated for assay determination. Assay (%w/w) of test solution at each level was calculated against three injections (n=3) of qualified Spiro alcohol reference or working standard in terms of Recovered (µg/ml) [Table 5.14]. Table 5.14: Recovery of the assay method for Spiro alcohol Spike level (%) Added (µg/ml) Recovered (µg/ml) Mean Recovery (%) %RSD The method have shown consistent and high recoveries at all three concentration (50%, 100% and 150%) levels which mean recovery ranging from 98.4% to 99.4%. The above accuracy/recovery study

25 201 indicated that the method was suitable for determination of Spiro alcohol Solution stability: The solution stability of Spiro alcohol in diluent in the assay method was performed by leaving the test solutions of sample in tightly capped volumetric flasks on a laboratory work table with room temperature for 48 hrs. The sample solution was assayed for every six hours interval upto the study time and freshly prepared reference standard was used each time to estimate the assay of sample. The %RSD of assay of Spiro alcohol during solution stability experiments were less than 0.1. The solution stability of Spiro alcohol in diluent in the related substances method was carried out by leaving the spiked test solutions of sample in tightly capped volumetric flasks at bench top, room temperature for 48hrs (two days). In test spiking solutions, the content of Imp-1, Imp-2, and Imp-3 are checked for every 12 hours interval up to the study period. No significant change was observed in the impurity content during solution stability experiments in the initial values up to study period. Hence Spiro alcohol spiked sample solution is stable for at least 48 hrs in the above developed method with the same diluent [Table Table5.16].

26 202 Table 5.15: Summary of related substances content obtained at different intervals & solution stability S.No. Interval Imp-1 Imp-2 Imp-3 Total impurities 1 0h 0.22% 0.15% 0.26% 1.11% 2 12h 0.22% 0.16% 0.26% 1.16% 3 24h 0.23% 0.16% 0.27% 1.16% 4 36h 0.23% 0.15% 0.27% 1.14% 5 48h 0.23% 0.16% 0.27% 1.15% Table 5.16: Summary of assay content obtained at different intervals & Solution stability S.No. Interval %Spiro alcohol 1 0h h h h h h h h h 99.7 Average 99.4 Stdev 0.14 %RSD 0.1

27 Mobile phase stability: The mobile phase stability of Spiro alcohol in diluent in the assay method was carried out by fresh test solutions of sample and mobile phase was kept constant up to 48hrs. The fresh same Spiro alcohol sample solutions were assayed for every six hours interval upto the study period, each time freshly prepared reference standard was used to estimate the assay of sample. The %RSD of assay of Spiro alcohol during mobile phase stability experiments were less than 0.1. The mobile phase stability of Spiro alcohol in diluent in the related substances method was carried out by spiked test solution leaving the mobile phase at room the temperature for 48 hours. Spiking solution containing Imp-1, Imp-2, and Imp-3 are checked for every twelve hours interval up to the study period. No major change was observed in the impurity content during mobile phase stability study experiments. Hence Spiro alcohol mobile phase solution is stable for at least 48 hrs in the above stated analytical method developed [Table Table 5.18]. Table 5.17: Summary related substances content obtained at different intervals & mobile phase stability results of the RS method S.No. Interval Imp-1 Imp-2 Imp-3 Total impurities 1 0h 0.23% 0.15% 0.25% 1.10% 2 12h 0.21% 0.17% 0.26% 1.15% 3 24h 0.23% 0.15% 0.26% 1.14% 4 36h 0.22% 0.16% 0.25% 1.12% 5 48h 0.25% 0.17% 0.25% 1.16%

28 204 Table 5.18: Summary assay content obtained at different intervals & mobile phase stability results of the assay method S.No. Interval %Spiro alcohol 1 0h h h h h h h h h 99.2 Average 99.3 Stdev 0.09 %RSD Robustness: To determine the robustness of developed method experimental conditions were intentionally altered and the resolution between critical pairs and USP tailing factor were evaluated in each deliberately altered chromatographic conditions [Table Table 5.20]. Table 5.19: System suitability-robustness Parameters Temperature Different flow Conditions The resolution between the impurity-3 (exo isomer) and TSC The tailing factor of TSC 22ºC ºC ºC ml ml ml

29 205 Table 5.20: Robustness-Relative retention time Parameters Conditions ~Relative Retention time Imp-1 Imp-2 Imp-3 Temperature 22ºC ºC ºC Different flow 0.5 ml ml ml Application of the Method to Analysis of Trospium chloride: As per USP monograph, the content of spiro alcohol in Trospium chloride is measured by using Thin layer chromatography (TLC). By the application of this validated method for the quantification of spiro alcohol, an accurate result can be obtained with in a short run time when compared to laborious process of TLC. 5.5 Summary and Conclusion: A simple, stereo selective HPLC-ELSD method developed for the separation of tropine, nortropine and exo isomer from spiro alcohol and quantitative determination of spiro alcohol is successfully validated. The present HPLC-ELSD method could be conveniently applied for the determination of spiro alcohol in Trospium chloride. [Table 5.21]

30 206 Table 5.21: Summary of the Validation Results Test parameter Related substances results Assay method Precision (%RSD) Imp-1 Imp-2 Imp-3 TSC LOD (µg/ml) LOQ (µg/ml) Intermediate precision Linearity Accuracy Rs between Rs between Rs between Robustness Exo isomer and TSC>1.5 Exo isomer and TSC>1.5 Exo isomer and TSC>1.5 NA Solution stability Stable up to 48hr Stable up to 48hr Stable up to 48hr Stable up to 48hr Mobile phase stability Stable up to 48hr Stable up to 48hr Stable up to 48hr Stable up to 48hr REFERENCES: [1] Product information: Sanctura TM, Trospium tablets, Esprit Pharmaceuticals, East Brunswick, NJ and Indevus Pharmaceuticals, Inc., Lexington, MA. [2] Fluorimetric determination of the quaternary compound trospium and its metabolite in biological material after derivatization with benoxaprofen chloride Gertrud Schladitz-Keil, Hildegard Spahn, E.

31 207 Mutschler, Journal of Chromatography B:Biomedical sciences and applications Volume345 pp [3] Evaporative light scattering detector: a new tool for screening purposes S.Cardenas, M.Gallego, M.Valcarcel, Anal. Chim. Acta 402(1999) 1-5. [4] A comparative study of commercial liquid chromatographic detectors for the analysis of underivatized amino acids Konstantinos Petritis, Claire Elfakir, Michel Dreux Journal of Chromatography A, 961 (2002) 9 21 [5] International Conference on Harmonization (ICH),. Q2 (R1), Validation of Analytical Procedures: Text and methodology [6] Validation of Compendial methods (2007), The United States Pharmacopeia, 30 th edn.usp30-