Acta Chim. Slov. 2003, 50, 321-326. 321 DETERMINATION OF ACETATE IN PHARMACEUTICAL PRODUCTS BY HPLCf Svjetlana Mihaljica, Jelena Trbojevič Institute ofPharmacy ofSerbia, Vojvode Stepe 458, Belgrade, Jugoslavia Marija Maskovič Institute ofPharmacy ofSerbia, Vojvode Stepe 458, Belgrade, Jugoslavia This paper is based on a presentation at The Fourth Slovenian Symposium on Separation Techniques meeting held in Novo Mesto, Slovenia during October 3-4th, 2002. Received 16-12-2002 Abstract HPLC method, using anion exchange column and UVTVis detector, for identification and determination of acetate in medicinal products is described in this study. SUPELCOSIL SAX1 strong anion exchange column has ion exchange character. In the ion exchange mode, different pH and ion strength will influence the retention tirne of acetate. Increasing the ionic strength will reduce its retention tirne. Changing the pH will control the protonation state of the analvte. Acetate has been found in those products as salt L-Lysinacetate. Validation of the method establishes that its performance characteristics (figures of merits, quality of parameters) are adequate for the intended use. It entails the evaluation of number of parameters, such as selectivity, linearity, accuracy, specificity, precision (repetability and reproducibility), sensitivity, detection and determination limits and robustness. Introduction Acetate was usually determined by titration with sodium hydroxide using phenolphtalein as indicator,1'4 or by non-aqueous titration with perchloric acid using/?-naphtholbenzein as indicator. In the meantime, the examinations of the content of acetate in pharmaceutical products as solutions for infusion by those methods had given the wrong results. Contents of secondary compounds, such as amino acids, in those products inhibit the accuracy of determining assay. There are references in the literature describing the identification, and determination of acetate in drug formulations using GC, HPLC, ' and Capillary Electrophoresis.6 S. Mihaljica, J. Trbojevič, M. Maškovič: Determination of Acetate in Pharmaceutical Products by HPLC 322 Acta Chim. Slov. 2003, 50, 321-326. The object of this study was to develop HPLC method, using anion exchange column and UV/Vis detector, for identification and determination of acetate in Aminoven and AMINO-MEL "nephro" solutions for infusion. Acetate has been found in those products as salt L-Lysinacetate. The proposed method needs to be fast, reliable, reproductive and used for daily routine laboratory work. Results and discussion The proposed HPLC method was developed and validated and could be used for identification and determination of acetate. The chromatographic process separates the two components (L-Lysine and acetate) from each other as well as from the excipients. SUPELCOSIL SAX1 strong anion exchange column has ion exchange character. In the ion exchange mode, different pH and ion strength will influence the retention tirne of acetate. Increasing the ionic strength will reduce its retention tirne. Changing the pH will control the protonation state of the analite. Acetate was identified by comparison of the retention times of the peak attributed to acetate in the sample solutions to that of standard solution. Cjuantification was achieved by comparing the peak area of acetate in sample with the corresponding peak in standard. A typical chromatograms containing L-Lysine and acetate were showed in Figure 1, Figure 2 and Figure 3. Efficiency, peak asymmetry and resolution establish chromatographic conditions that ensure the system in a manner suitable to carry out the analysis. Efficiency was confirmed calculating the number of theoretical plates (N) for the chromatographic column using the acetate peak. The number of theoretical plates was 5827 (>2000). Peak asymmetry was confirmed calculating peak asymmetry (T) for the acetate peak. Peak asymmetry was 1.5 (<=1.5). Resolution was confirmed calculating the resolution (R) betvveen the L-Lysine and acetate peaks. The resolution was 14.2. Estimates of limits of detection and quantitation for acetate were obtained from the linearity data. Based on the average of the three founded peak area values, limits of detection is 8.47-10"5 mol/L and limits of quantification is 1.69-10"4 mol/L. S. Mihaljica, J. Trbojevič, M. Maškovič: Determination of Acetate in Pharmaceutical Products by HPLC Acta Chim. Slov. 2003, 50, 321-326. 323 Solutions of acetate encompassing 80-120% of the target level (1.7-10° mol/L) were analyzed in absence of excipient blend. A plot of peak area versus concentration in absence of excipient blend is shown in Figure 4. The HPLC method was shown to be linear over the concentration (1.36-10" -2.04-10" mol/L), y — 1E+06 x -99767, R2=0.9997. 250 200 150™ A 100 50 250 200 L m150 A i! -A U \ 100 \ 50 [_____________ v, 1 —^-^ ) 5 Minutes 10 15 Figure 1. A typical HPLC Profile of L-Lysinacetate Working Standard Solution. 250 200 m150 A U 100 50 \ 250 200 150™ A 100 50 0 5 Minutes 10 15 Figure 2. Chromatogram of Sample Solution AMINO-MEL "nephro" (batchno. BI20024). 250= 200' m150> A U 100= 50 6 VJ 250 200 150™ A 100 50 5 Minutes 10 Figure 3. Chromatogram of Sample Solution Aminoven 5 % (baten no. MG 1596). 15 S. Mihaljica, J. Trbojevič, M. Maškovič: Determination of Acetate in Pharmaceutical Products by HPLC 324 Acta Chim. Slov. 2003, 50, 321-326. 2.9 n 2.7 o 2.5 o o § 2.3 o x 2.1 < 1.9 1.7 1.5 y = 1 E+06x -99767 R2 = 0.9997 1.15 1.35 1.55 1.75 1.95 Acetate concentration (mol/L x 10") Figure 4. Peak Area vs. Concentration for Acetate in absence of placebo matrht. The HPLC system precision was determined at the assay target lvel by performing seven replicate injections of a solution containing 1.7-10" mol/L acetate. A relative standard deviation of 0.99% based on peak area was obtained. The data are presented in Table 1. Table 1. Acetate System Precision. Replicate 1.7-10" 3mol/L Peak Area 1 2283349 2 2238358 3 2250278 4 2273199 5 2239980 6 2268940 7 2297456 Mean: 2264509 Standard Deviation: 22448.7 % Relative Standard Deviation: 0.991 The accuracy of the method was tested by measuring the recovery of an acetate. Nine determinations for concentration 80%, 100% and 120% of the target level (1.7-10"" mol/L) utilized, with quantification by peak area. The average recoveries were 99.7%, S. Mihaljica, J. Trbojevič, M. Maškovič: Determination of Acetate in Pharmaceutical Products by HPLC Acta Chim. Slov. 2003, 50, 321-326. 325 with a relative standard deviation of 1.60%, for concentration 1.36-10" mol/L (80%), 99.6%, with a relative standard deviation of 0.69%, for concentration 1.7-10" mol/L (100%>) and 99.3%, with a relative standard deviation of 1.27%, for concentration 2.04-10" mol/L (120%>). The data are presented in Table 2. Table 2. Accuracv- Recovery of Acetate. mol/L Added mol/L Found 1 % Recoverv 1.36-10"3 1.34-10"3 98.46 1.36-10"3 1.35-10"3 99.13 1.36-10"3 1.38-10"3 101.50 Mean: 99.7 Standard deviation: 28306.4 % Relative Standard Deviation: 1.60 1.7-10"3 1.70-10"3 100.02 1.7-10"3 1.70-10"3 99.97 1.7-10"3 1.68-10"3 98.81 Mean: 99.6 Standard deviation: 15377.5 % Relative Standard Deviation: 0.69 2.04-10"3 2.05-10"3 100.67 2.04-10"3 2.05-10"3 99.04 2.04-10"3 2.00-10"3 98.19 Mean: 99.3 Standard deviation: 34208.3 % Relative Standard Deviation: 1.27 Conclusions The method for determining the content of acetate according to the above mentioned subject is validated. The specificitv, accuracv, linearitv, and the precision of the method were tested. In addition, the quantification and detection limit of acetate were determined. Based on the validation data, the described procedure is suitable for the identification and determining the content of acetate in Aminoven and AMINO-MEL "nephro " solutions for infusion. S. Mihaljica, J. Trbojevič, M. Maškovič: Determination of Acetate in Pharmaceutical Products by HPLC 326 Acta Chim. Slov. 2003, 50, 321-326. Experimental HPLC analyses were perfomed with a GBC system LC 1120 HPLC Pump equipped with an LC 1210 UV/Vis detector and Rheodyne injector valve (20|aL sample loop). Separation was made on a Supelcosil LC-SAX1 column (250 x 4.6 mm i.d., particle size 5\im), using mobile phase acetonitrile-water (3:97 V/V) at pH 3.0 adjusted with H3PO4. The flow rate was 2.0 ml/min. The detector wavelength was 215 nm, range 0.01 AUFS. Reference solutions contain 0.28 - 0.42 mg/mL L-Lysinacetate in mobile phase, equivalentto 0.08 -0.12mg/mL (1.36103- 2.04-10"3 mol/L) acetate. Test solutions are prepared by diluting the measured amounts of examined solutions with the mobile phase and filtering through 0.45 um PTFE membrane filters. A 20|aL aliqout of each examined solution was injected in triplicate. Ali measurements were based on single injections. Since peak area measurements provided better perfomance than peak height, it was chosen for use in test procedure. Acknowledgements This work was supported by the Institute of Pharmacy of Serbia, Belgrade. References and Notes 1. Pharmacopeia Jugoslavica 2000, editio quinta, Savremena administracija, Beograd. 2. U.S. Pharmacopeia 25, NF 20, Board of Trustees 2002 42, 855, 1574. 3. British Pharmacopeia 1998. The Stationery office, London, Appendix VIA174. 4. European Pharmacopoeia, Fourth Edition 2002, 576. 5. T. K. Natishan, and P. Sajonz, J. Liq. Chromatogr. Relat. Technol. 2001, 24, 2583-2599. 6. T. Soga, M. Wakaura, J. Am. Soc. Brew. Chem. 1997, 44-46. 7. lonpac® ICE-AS1 ion exclusion column, Dionex Corporation, 1996, Figure 1. Povzetek Za identifikacijo in določitev acetata v medicinskih produktih smo uporabili HPLC z anionsko izmenjevalno kolono in UV7VIS detektorjem. Na retenzijski čas vplivajo pH in ionska moč. Povečanje ionske moči na primer zmanjša retenzijski čas. S. Mihaljica, J. Trbojevič, M. Maškovič: Determination of Acetate in Pharmaceutical Products by HPLC