Original Article 321
Authors
B. K. Chaursia, T. P. Singh, B. Varshney, P. Sharma, S. S. Iyer, A. H. Khuroo, T. Monif
Affiliation
Metabolism and Pharmacokinetics, Department of Clinical Pharmacology & Pharmacokinetics, Ranbaxy Laboratories Limited, Haryana, India
Key words ▶ meropenem ● ▶ solid phase extraction ● ▶ validation ● ▶ rat plasma ● ▶ dose proportionality study ● ▶ LC-MS/MS ●
Abstract
received 01.06.2013 accepted 16.10.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1358702 Published online: November 13, 2013 Drug Res 2014; 64: 321–329 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S. S. Iyer Department of Clinical Pharmacology & Pharmacokinetics Ranbaxy Laboratories Limited Plot No. GP-5 HSIDC Sector-18 Old Delhi-Gurgaon Road Gurgaon-122015 Haryana India Tel.: + 91/124/4768 016 + 91/124/4768000 Fax: + 91/124/4231 002 [email protected]
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A simple, rapid, sensitive and selective assay based liquid chromatography-tandem mass spectrometric method was developed and validated for quantitative analysis of meropenem in rat plasma using rolipram as internal standard. Efficient chromatographic separation of analyte from matrix components was achieved by using Kromasil 100 C18 (150 × 4.6 mm, 5 μm) reversed phase column with mobile phase consisting of acetonitrile and 2 mM ammonium acetate buffer (80:20, v/v) delivered in isocratic mode with constant flow rate of 0.7 mL/min. Detection of meropenem and rolipram was performed in positive mode using multiple reaction monitoring (MRM). The mass transitions 384.1→141.0 and 276.2→191.1 were used to quantify meropenem and rolipram, respectively. A fast and simple
Introduction
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Meropenem {chemically known as -[5-(dimethylcarbamoyl) pyrrolidin-2-yl] sulfanyl6- (1-hydroxyethyl)-4-methyl-7-oxo- 1-azabicyclo[3.2.0] hept-2-ene-2-carboxylic acid} is an ultra-broad spectrum injectable antibiotic used for treatment of wide variety of infections such as meningitis, urinary tract, febrile neutropenia, ▶ Fig. 1). pneumonia and cystic fibrosis [1–4] (● Meropenem was originally developed by Sumitomo Pharmaceuticals, but after approval by USFDA in 1996 it is currently available globally with brand names such as Merrem, Meronem, Zwipen, Mepem, Meropen and Neopenem etc. Meropenem is a high molecular weight drug (Mw 383.46) with poor and pH dependent aqueous solubility. It has 2 pKa values of 2.9 and 7.4 [5]. Meropenem is generally given by intravenous administration and supplied as white crystalline powder, to be dissolved in 5 % (w/v) potassium phosphate solution for intravenous administra-
solid phase extraction method was optimized for extraction of meropenem from rat plasma. The developed method was validated for selectivity, accuracy, precision, linearity, recovery, stability, matrix effect, dilution integrity as per regulatory guidelines. The developed method was selective with no interfering components at the retention time of meropenem and rolipram. The assay demonstrated acceptable linearity (R2 > 0.99) over a dynamic range of 0.19–201.40 μg/mL. The method exhibited excellent and acceptable precision and accuracy, and produced consistent recoveries. The method demonstrated excellent stability of meropenem in rat plasma under studied conditions investigated. Finally, the validated method was successfully applied to quantify meropenem levels in rat plasma of a dose escalation study.
tion. In humans, upon intravenous administration of 1 g of meropenem, Cmax levels up to 30 mg/ lit were observed. The elimination half life of meropenem was found to be 1 h with volume of distribution up to 21 lit. The Cmax and AUC of meropenem increase linearly with dose indicating the absence of saturation phenomenon [6]. It exhibits negligible plasma protein binding (up to 2 %) and penetrates well into most of body tissues including cerebrospinal fluid, heart and lung muscles, bile and peritoneal fluids. Even though the pharmacokinetics of meropenem in humans is extensively studied, the information related to disposition in laboratory animals such as rat is very limited. In addition, estimation of pharmacokinetics of meropenem in laboratory animals is essential during the safety assessment of toxicity of drug formulations in humans. Hence the objective of the present work was to develop a simple and efficient bioanalytical method for the quantification of meropenem in rat plasma.
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Development and Validation of Liquid Chromatography-Tandem Mass Spectrometric Method for Quantification of Meropenem in Rat Plasma and its Application in a Preclinical Dose Proportionality Study
322 Original Article
generation of charged ions in multiple reactions monitoring mode. The HPLC system (Perkin Elmer, Shelton, USA) consisting of PE 200 series autosampler, PE 200 series pump were used for injections of analyte solutions on to LC-MS/MS. The temperature of autosampler vial tray was maintained at 6 °C. The optimized mobile phase consisted of acetonitrile and 2 mM ammonium acetate (80:20, v/v) delivered at a flow rate of 0.7 mL/min. Upon 5 μL of injection on to a Kromasil 100 C18 reversed phase column (150 × 4.6 mm, 5 μ, Grace Davison, USA) the column eluents were monitored in mass spectrometer using transitions of 384.1→141.0 and 276.2→191.1 for meropenem and rolipram, respectively. The chromatograms were acquired using Analyst® software (Ver 1.4.1, Applied Biosystems, MDS SCIEX, Toronto, Canada).
Literature review revealed many high performance liquid chromatographic (HPLC) methods for the quantification of meropenem in pharmaceutical formulations and in biomatrices such as plasma, serum and bile [7–14]. The reported methods are time consuming, laborious and involved complex techniques such as online micro dialysis and column switching. In contrast to HPLC, modern liquid chromatography-tandem mass spectrometric (LC-MS/MS) methods offer ultra-fast analysis and involve minimal sample preparation. Only 2 LC-MS/MS methods have been reported for quantification of meropenem in cerebrospinal fluid and human plasma. The injection volumes used for quantification were upto 50 μL in published literature while 5 μL injection volume was used in present work [15, 16]. In the present work, with a lower limit of quantification, a simple and efficient LC-MS/MS method was developed and fully validated for quantification of meropenem for the first time in rat plasma. The method was applied for estimation meropenem in plasma samples of a dose escalation in rats.
Experimental
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Chemicals & reagents Meropenem (assay ≥ 87.6 %) working standard was supplied by Analytical Research Division of Ranbaxy Laboratories Limited, India. Rolipram (purity ≥ 98 %) was used as internal standard and procured from Sigma Aldrich, USA. HPLC grade acetonitrile and methanol were purchased from S.D. Fine Chemicals Ltd., Mumbai, India. Ammonium acetate and acetic acid were obtained from Sigma Aldrich, USA. Sodium bicarbonate and sodium citrate were purchased from Ranbaxy Fine Chemicals, New Delhi, India. Oasis HLB solid phase extraction cartridges (30 mg, 1 mL) were purchased from Waters corporation, Massachusetts, USA. Milli Q water (18.2 mΩ & TOC ≤ 50 ppb) from a Milli-Q® water purification system (Millipore SAS, Molshem, France) was used. All other chemicals and reagents used in the study were of ARgrade. The normal batches of Wistar Hannover rat plasma for method validation were obtained from in-house facility; while lipemic and hemolyzed batches were procured from Bioreclamation incorporation, Newyork, USA.
Instrumentation The quantification of meropenem in rat plasma was performed on API-4000 LC-MS/MS system (Applied Biosystems, MDS SCIEX, Toronto, Canada) coupled with electrospray ionization used for
Animals All the study protocols were approved by Institutional Animal Ethics Committee (IAEC, protocol: 68/08), Ranbaxy and were in accordance with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and OECD principles for Good Laboratory Practices (GLP). Healthy male Wistar Hannover rats with weight range of 130–250 g (4–7 weeks old) were obtained from Animal Breeding and Housing Facility, Ranbaxy Laboratories Limited, Gurgaon. After screening, the animals were placed in polycarbonate cage in groups of 4 and maintained in controlled environment conditions. Animals were acclimatized 7 days prior to commencement of work and they were provided autoclaved, standard pelleted laboratory animal diet, with free access to purified water.
Preparation of calibration curve and quality control samples The stock solution of meropenem was prepared by dissolving 20 mg of working standard in 1 mL of 0.5 % (w/v) sodium bicarbonate solution. Primary stock solution of meropenem for the preparation of calibration curve standard and quality control samples (QC) were prepared separately. Subsequent dilutions for calibration curve standard and quality control samples were prepared in 0.5 % (w/v) sodium bicarbonate solution. Spiking of aqueous dilutions from primary stock solution was done in plasma to get 8 point calibration curve (0.19–201.40 μg/mL) of meropenem. 8 non-zero of calibration curve standards were prepared by spiking 50 μL of each working stock into 950 μL of blank rat plasma to achieve final concentration of 0.19, 0.47, 1.18, 4.72, 18.88, 75.53, 151.05, 201.40 μg/mL. Quality control samples were prepared independently at 5 concentration level. These included the lower limit of quantification (LLOQQC, 0.19 μg/mL), within 3 times of lower limit of quantification (LQC, 0.48 μg/mL), in the middle (MQC, 80.48 μg/mL), near upper concentration (HQC, 160.96 μg/mL) and above upper concentration (DQC, 402.41 μg/mL). The aliquot of 75 μL of each CC & QC samples was stored below − 50 °C until analysis. The primary stock solution for internal standard (IS) was prepared in acetonitrile to obtained final concentration of 1.0 mg/mL. An IS working solution of 10.0 μg/mL was prepared by diluting primary stock with water/acetonitrile 50/50, v/v.
Sample preparation Solid phase extraction was used for extraction of drug from plasma. For sample preparation, 50 μL of plasma sample was mixed with 50 μL internal standard solution (Rolipram 10 μg/ mL) and 500 μL of 2 mM ammonium acetate buffer (pH adjusted
Chaursia BK et al. Method for Quantification of Meropenem … Drug Res 2014; 64: 321–329
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Fig. 1 Chemical structure of meropenem.
Original Article 323
Method development Electrospray (ESI) was selected as an ionization source in the present study. For tuning purpose, the standard solutions of meropenem and rolipram ( ≈ 250 ng/mL) were prepared in mixture of acetonitrile and MlliQ water (50:50, v/v) and were infused. Both analyte and internal standard were optimized in both positive and negative mode for obtaining maximum intensity. The parent and product ions for multiple reaction monitoring (MRM) were selected after scanning in Q1 MS, Q3 MS, product ion and precursor ion modes. After selection of mass transitions for MRM, various voltages such as declusterization potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) and collision activated dissociation (CAD) gas were optimized to produce maximum intensity. Other mass spectrometric parameters viz. nebulizer gas, heater gas, curtain gas, temperature, ion spray voltage were optimized by flow injection analysis (FIA). The separation of meropenem was achieved by liquid chromatographic technique by optimizing combination of water, various buffer solutions, acetonitrile and methanol. The peak properties of meropenem were optimized by selection of suitable reversed phase column. Other chromatographic parameters like flow rate and injection volume were optimized for better sensitivity, good resolution, as well as shorter run time for analyte and IS. Several internal standards were tried; however chromatography, consistent response and reproducibility were obtained with rolipram. Therefore rolipram was selected as internal standard. Following optimization of mass spectrometer condition, each step in processing, such as sample preparation, chromatographic separation of interfering components from analyte peak, extraction efficiency were optimized for developing a sensitive and reproducible bioanalytical method. Various sample clean up techniques such as protein precipitation using methanol and acetonitrile, liquid-liquid extraction using various organic solvents such as ethyl acetate, tertiary methyl butyl ether, dichloromethane, n-hexane alone and in combination were evaluated for maximizing extraction efficiency. Finally solid phase extraction using various sorbents and various combinations of washing and eluting solvents was performed for enhanced sample clean-up and extraction efficiency.
Method validation The developed bioanalytical method for quantification of meropenem in rat plasma was validated with respect to various parameters such as selectivity, linearity and range, sensitivity, accuracy and precision, carry over, recovery, matrix effect, stability of meropenem in rat plasma under various conditions, dilution integrity and reinjection reproducibility as per USFDA guidelines for bioanalytical method validation [17].
Application of bioanalytical method to a dose escalation study in rat The dose escalation study in rat was performed by administering meropenem at various dose levels such as 500, 1 000 and 2 000 mg/kg by intravenous route through caudal vein. After a predefined inclusion and exclusion criteria, total of 63 animals were selected and divided into 3 groups of 21 for each dose level. The formulations were prepared by dissolving meropenem in dextrose solution (10 %, w/v) to achieve 50, 100 and 200 mg/mL solutions and were used immediately to dose 500, 1 000 and 2 000 mg/kg respectively. The dose volume was 10 mL/kg of the body weight of animal. The administration was done at the rate of 2 mL/min using a calibrated infusion pump (Harvard Apparatus, Mumbai, India). Approximately, 250 μL of blood samples were collected from retro-orbital plexus at 0.083, 0.25, 0.5, 1.0, 1.5, 2.0, 4.0 h in an Eppendorf tube containing 10 μL of sodium citrate solution (20 %, w/v). 3 rats were used for a single time point and the blood samples were immediately placed on wet ice. The collected blood samples were centrifuged (at 10 000 rpm, 4 °C for 5 min) within 30 min of collection to separate plasma and stored immediately below − 50 °C until analysis. The concentration-time data was analyzed using validated WinNonlin Professional software (Version 5.0.1, Pharsight Corporation, Cary, North Carolina) and the pharmacokinetic parameters such as Cmax, T1/2, AUC0–t and AUC0–∞ were estimated by non-compartmental approach at each dose level. The Cmax is calculated as the highest concentration observed in the profile. The elimination rate constant (λz) was obtained from the slope of linear regression line of log transformed concentration vs. time profile. The elimination half life is calculated from λz using the expression 0.693/λz. The area under the curve till last measurable concentration (AUC0–t) was calculated from log-linear trapezoidal rule. The area under curve extrapolated to infinity (AUC0–∞) was calculated from the expression AUC0–t + Ct/λz, where Ct is the last measurable concentration.
Results and Discussion
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Method development Depending on the polarity and molecular weight of meropenem and rolipram, ESI was selected as an ionization source in the present study. Both the molecules showed maximum intensity in Q1MS in positive ion mode in comparison to negative mode and it was selected for further optimization. In Q1MS scan mode the, most abundant protonated ion [M + H] + for meropenem ▶ Fig. 2), and rolipram (276.2) were selected. In the (m/z 384.1) (● product ion mode, the values of CE and CAD were varied and optimized for selection of most abundant product ion for mero▶ Fig. 3) and rolipram (191.1). Moreover, the penem (141.0) (● parent ions for selected product ions were confirmed by scanning in precursor mode for both meropenem and rolipram. After the selection of parent to product ion transition, various MS parameters like DP, EP and CXP were optimized for meropenem and rolipram in MRM mode. The optimized values of MS param▶ Table 1. After eters for meropenem and rolipram are given in ● the optimization of MS parameters, chromatographic optimization was performed aiming at better sensitivity and superior peak properties. Experiments carried out with combination of water and methanol (50:50, %v/v) using a C18 reversed phased column showed peak tailing for meropenem. Replacing methanol
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to 4.8 with glacial acetic acid) and vortexed for homogeneous mixing. The content was transferred to Oasis HLB extraction cartridge preconditioned with 1 mL methanol and 1 mL of 2 mM ammonium acetate buffer (pH 4.8) at a constant pressure of 10–15 psi. The loaded cartridge was washed with 1 mL of 2 mM ammonium acetate buffer (pH 4.8) and 1 mL of MilliQ water to remove matrix components. Finally, the elution was carried out into a clean glass tube twice with 700 μL of elution solution (acetonitrile/2 mM ammonium acetate buffer (pH 4.8, 50:50, v/v). The samples were vortexed for homogeneity and 5 μL was injected on to LC-MS/MS for analysis.
324 Original Article
Fig. 3 Product ion mass spectrum of meropenem for selection of product ion.
with acetonitrile resulted in improved peak properties for both meropenem and rolipram but resulted in poor retention. Further optimization of peak properties was carried out by incorporation of buffering agents such as ammonium acetate (2 mM) into the mobile phase. Among all combinations of acetonitrile
and 2 mM ammonium acetate, 80:20 ( %v/v) showed better peak parameters and improved retention for both meropenem and rolipram. Further optimization of retention was carried out by optimizing the flow rate to 700 μL/min and injection volume of 5 μL was selected based on the signal to noise ratio (S/N) in
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Fig. 2 Q1MS spectrum of meropenem for selection of parent ion.
Original Article 325
Table 1 Optimized MS parameters for meropenem and rolipram. MS Parameter
Meropenem
Rolipram
Transition (m/z) DP (Volt) EP (Volt) CE (Volt) CXP (Volt) Nebulizer gas (Psi) Heater gas (Psi) Temperature ( °C) Ion spray voltage (Volt)
384.1/141.0 85 10 22 8
276.2/191.1 80 10 24 8 50 50 500 5 500
Method validation Selectivity Chromatograms of blank samples revealed no interfering peaks eluting at the retention time of meropenem or rolipram. Lack of response from endogenous impurities resulting from biomatrix indicated the selectivity of the method for quantification of meropenem from rat plasma. Comparison of chromatograms of blank sample, zero sample and LLOQ sample confirmed the ▶ Fig. 4–6). In addition, analysis of selectivity of the method (● blank and LLOQ samples from hemolyzed and lipemic plasma resulted in no significant effect of endogenous impurities on the quantification of meropenem, indicating the applicability of method for various types of plasma samples.
Linearity The linear regression analysis indicated the linear relationship between meropenem concentration and area ratio (meropenem to rolipram) over a dynamic range of 0.19–201.4 μg/mL. The coefficient of linear regression (R2) values was greater than 0.99 in all analytical batches indicating the linearity of proposed method. Weighting factor 1/x2 was chosen based on the concentration-response relationship. The best fit linear regression equation estimated from linear regression analysis was: peak area ratio (Analyte/IS) = 0.0174 ± 0.0021 × Concentration of meropenem (μg/mL) + 0.0000943 ± 0.0001. The calculated intercept was significantly low indicating the accuracy of the method and insignificant contribution of matrix components in the quantification of meropenem.
Precision and accuracy The obtained results for %CV and %nominal represent the precision and accuracy of the proposed method as the values were in accordance with the regulatory acceptance criteria. The results ▶ Table 2a, b. The overall of precision and accuracy are shown in ● within precision and accuracy ranged from 1.93–11.29 % and 93.80–104.24 %, respectively. The intra-day precision and accuracy values ranged from 2.46–9.52 % and 93.80–103.28 %, respectively. In addition, the inter-day precision accuracy ranged from 2.90–8.20 % and 95.91–101.73 %, respectively. The extended
Fig. 4 Representative chromatogram for blank rat plasma.
Chaursia BK et al. Method for Quantification of Meropenem … Drug Res 2014; 64: 321–329
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processed LLOQ samples. Based on all the above trials, the optimized mobile phase combination consisting of acetonitrile and 2 mM ammonium acetate (80:20, %v/v) and the retention times of meropenem and rolipram in this combination were 1.5 and 2.5 min, respectively over a run time of 3 min. For sample preparation, a single step precipitation with methanol or acetonitrile resulted in insufficient sample clean up and high protein load resulting in increasing back pressure of the column. Liquid liquid extraction with ethyl acetate, tertiary methyl butyl ether, dichloromethane, n-hexane alone and in various combinations showed poor extraction efficiency for meropenem. Acidification of plasma showed marginal increase in recovery for meropenem. In order to increase extraction efficiencies, produce a cleaner chromatogram, overcome the problems of high back pressure and improper peak shape, solid phase extraction was evaluated for the extraction of meropenem from plasma. Solid phase extraction using Oasis HLB cartridges provided better recovery as compared to other cartridges. As meropenem is weakly acidic (pKa 2.9, 7.4), the conditioning and washing steps during solid phase extraction were optimized with 2 mM ammonium acetate (pH, 4.8) for better retention of meropenem on the solid phase sorbent. Finally, the elution of meropenem was carried out using a combination of acetonitrile and 2 mM ammonium acetate (pH, 4.8) (50:50, v/v) for better recoveries from rat plasma.
326 Original Article
Fig. 6 Representative chromatogram for lower limit of quantification.
batch precision and accuracy values ranged from 4.97–7.30 % and 91.09–99.64 %, respectively indicating the precision and accuracy of the proposed method over an analytical batch consisting of 120 samples.
Carryover effect The method demonstrated no carryover for meropenem and rolipram as there was no observed peak area for meropenem and rolipram in any of the 2 blank samples injected after the upper limit of quantification samples.
Sensitivity The plasma standards prepared at LLOQ level were analyzed in 5 replicates in 3 analytical batches. The comparison of chromato▶ Fig. 6) with blank sample (● ▶ Fig. 4) indicate gram of LLOQ (● the sensitivity of the method as the obtained S/N ratio was significantly higher than 5. The between batch precision and accuracy values at LLOQ in 3 analytical batches were 5.00 % and 105.26 %, respectively demonstrating the sensitivity of the method for quantification of meropenem.
Matrix effect The method demonstrated insignificant matrix effect from endogenous impurities in the quantification of meropenem. The matrix factor values obtained during the method development for meropenem and rolipram were 0.98 ± 0.0298 and 0.98 ± 0.0666, respectively indicating the negligible contribution of matrix components in the quantification of meropenem. Moreover, the value of IS normalized matrix factor was 1.009 ± 0.040 indicating the similar matrix effect for meropenem and rolipram. The obtained within batch precision and accuracy values during validation from 12 sets of LQC and HQC ranged from 1.16–4.46 % and 95.68–104.34 %.
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Fig. 5 Representative chromatogram for blank rat plasma spiked with rolipram.
Original Article 327
QC
LLOQ QC
LQC
MQC
HQC
Nominal Concentration (μg/mL) Batch-1 Mean SD CV %nominal N Batch-2 Mean SD CV %nominal N Batch-3 Mean SD CV %nominal N Extended batch Mean SD CV %nominal N
0.19
0.48
80.48
160.96
0.19 0.02 11.29 101.05 5
0.46 0.01 1.93 96.67 5
82.34 5.27 6.40 102.31 5
156.59 5.08 3.24 97.29 5
0.19 0.01 3.72 100.00 5
0.48 0.01 2.29 98.58 5
83.90 4.53 5.40 104.24 5
155.55 6.21 3.99 96.64 5
0.19 0.02 9.52 98.95 5
0.47 0.02 3.82 97.50 5
79.38 4.76 5.99 98.63 5
150.97 3.71 2.46 93.80 5
0.44 0.02 5.41 91.09 40
80.19 5.86 7.30 99.64 40
153.33 7.62 4.97 95.26 40
LLOQ QC
LQC
MQC
HQC
Nominal Concentration (μg/mL) Within Batch Mean SD CV %nominal N Intra-day (Batch 1&2) Mean SD CV %nominal N Intra-day (Batch 3) Mean SD CV %nominal N Inter-day Mean SD CV %nominal N
The absolute recoveries of meropenem and rolipram were calculated by analyzing the 5 replicates of LQC, MQC and HQC and comparing against freshly prepared aqueous samples. The proposed method produced consistent absolute recoveries at all studied levels such as LQC, MQC and HQC, respectively. The overall recovery for meropenem and rolipram were calculated to be 38.39 ± 5.82 and 121.11 ± 8.54, and %CV were 15.17 & 7.05 respectively. The precision at LQC, MQC and HQC levels were 3.80, 6.01 and 5.06, respectively. The consistent recoveries indicate that the developed method was precise and accurate for quantification of meropenem in rat plasma.
Stability ▶ Table 3. The results The result of stability exercises is given in ● demonstrate that meropenem is stable under various storage and processing conditions. No significant degradation of meropenem was observed upon 3 freeze thaw cycles as the mean stability ranged from 92.92–97.97 % and 93.33–97.83 %, respectively for 1 and 3 freeze thaw cycles. The LQC and HQC samples stored at room temperature over wet ice demonstrated the stability of meropenem as the mean accuracy ranged from 102.34–103.33 % after 2 h. Long term stability study showed that meropenem is stable in rat plasma up to 39 days when stored below − 50 °C as there was no significant change in response when compared with 0 h samples. The mean accuracy values for meropenem up to 39 days ranged from 99.17–100.76 %. The processed LQC and HQC samples were stable in autosampler conditions up to 24 h as the mean accuracy values for meropenem and rolipram ranged from 93.35–94.58 % and 98.21–104.95 %, respectively.
Dilution integrity
Table 2b Precision and accuracy data for meropenem in rat plasma. QC
Recovery
0.19
0.48
80.48
160.96
0.19 0.01 3.72 100.00 5
0.48 0.01 2.29 99.58 5
83.90 4.53 5.40 104.24 5
155.55 6.21 3.99 96.64 5
0.19 0.02 7.98 100.53 10
0.47 0.01 2.54 98.13 10
83.12 4.70 5.66 103.28 10
156.07 5.37 3.44 96.96 10
0.19 0.02 9.52 98.95 5
0.47 0.02 3.82 97.50 5
79.38 4.76 5.99 98.63 5
150.97 3.71 2.46 93.80 5
0.19 0.02 8.20 100.00 15
0.47 0.01 2.90 97.92 15
81.87 4.90 5.99 101.73 15
154.37 5.35 3.47 95.91 15
The developed method demonstrated over curve dilution integrity for 10, 20 and 40-folds dilution with rat plasma. The within batch accuracy values for 10, 20 and 40-folds dilution were 107.34, 95.72 and 103.60 %, respectively.
Reinjection reproducibility The reinjection reproducibility of the method was evaluated to demonstrate the integrity of meropenem upon piercing the sample closure. The results demonstrated reinjection reproducibility of meropenem up to 3 injections as the %initial with respect to first injection ranged from 92.31–102.28 % at LQC and HQC.
Application of bioanalytical method to a dose escalating study Plasma concentrations of meropenem in rat declined monoexponentially and were below the limit of quantitation by 4 h post dose. The mean plasma concentration vs. time profiles of mero▶ Fig. 7, 8. The penem at all studied dose levels are overlaid in ● mean pharmacokinetic parameters of the parent compound are ▶ Table 4. The exposures (AUC outlined in ● 0–t) increased proportional to the dose (R2 > 0.95) and were found to be 232.92, 314.69, 940.10 h.μg/mL respectively following 500, 1 000 and 2 000 mg/kg doses. AUC0–t at all dose levels were more than 95 % of AUC0–∞ indicating that the samples were collected long enough to determine total exposure from the doses administered. Like other penams, meropenem is also cleared very rapidly in the rat with short half life and small volume of distribution (Vd).
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Table 2a Precision and accuracy data for meropenem in rat plasma (Individual batches).
328 Original Article
QC Sample
Stability
Calculated conc. (μg/mL)*
LQC HQC
Freeze-thaw (1 cycles)
LQC HQC
Freeze-thaw (3 cycles)
LQC HQC
Bench top (2 h in wet ice)
LQC HQC
Autosampler (24 h)
LQC HQC
Long term (39 days)
0.45 ± 0.01 157.69 ± 2.36 0.45 ± 0.01 157.47 ± 1.44 0.50 ± 0.02 164.72 ± 1.05 0.45 ± 0.01 150.26 ± 2.72 0.48 ± 0.02 162.18 ± 0.48
% CV
% nominal
2.01 1.50 2.91 0.91 2.01 1.50 1.97 1.81 3.52 2.95
92.92 97.97 93.33 97.83 103.33 102.34 94.58 93.35 99.17 100.76
Table 3 Stability data for meropenem in rat plasma.
*each value is average of 5 determinations and results are expressed as Mean ± SD
Conclusion In the present work, a new, simple LC-MS/MS method was developed and validated for quantitative determination of meropenem from rat plasma. The method was fully validated for various method validation parameters as per regulatory guidelines. Simple and fast solid phase extraction resulted in consistent and reproducible recoveries of meropenem over the dynamic range. The method was found to be accurate, precise with excellent stability of meropenem under various processing and storage conditions. Finally, the method was successfully employed for determining the dose proportionality of meropenem upon intravenous administration in rats. Fig. 7 Mean plasma concentration vs. time profile of meropenem upon intravenous administration at all studied dose levels.
Conflict of Interest
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Authors of this publication do not have any Conflict of Interest.
References
Fig. 8 Semi log plot of mean plasma concentration vs. time profile of meropenem upon intravenous administration at all studied dose levels.
Table 4 Mean pharmacokinetic parameters of meropenem in rats upon intravenous administration. Dose
Tmax
Cmax
AUC0–t
AUC0–∞
Cl
(mg/kg)
(h)
(μg/mL)
(h.μg/mL)
(h.μg/mL)
(mL/h/kg) (mL/kg)
500 1 000 2 000
0.083 836.24 232.92 0.083 1 127.47 314.69 0.083 3 363.33 940.10
233.24 315.10 940.46
2 143.67 3 173.55 2 126.60
Vd 287.61 434.08 290.33
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Chaursia BK et al. Method for Quantification of Meropenem … Drug Res 2014; 64: 321–329
Authors
B. K. Chaursia, T. P. Singh, B. Varshney, P. Sharma, S. S. Iyer, A. H. Khuroo, T. Monif
Affiliation
Metabolism and Pharmacokinetics, Department of Clinical Pharmacology & Pharmacokinetics, Ranbaxy Laboratories Limited, Haryana, India
Key words ▶ meropenem ● ▶ solid phase extraction ● ▶ validation ● ▶ rat plasma ● ▶ dose proportionality study ● ▶ LC-MS/MS ●
Abstract
received 01.06.2013 accepted 16.10.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1358702 Published online: November 13, 2013 Drug Res 2014; 64: 321–329 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S. S. Iyer Department of Clinical Pharmacology & Pharmacokinetics Ranbaxy Laboratories Limited Plot No. GP-5 HSIDC Sector-18 Old Delhi-Gurgaon Road Gurgaon-122015 Haryana India Tel.: + 91/124/4768 016 + 91/124/4768000 Fax: + 91/124/4231 002 [email protected]
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A simple, rapid, sensitive and selective assay based liquid chromatography-tandem mass spectrometric method was developed and validated for quantitative analysis of meropenem in rat plasma using rolipram as internal standard. Efficient chromatographic separation of analyte from matrix components was achieved by using Kromasil 100 C18 (150 × 4.6 mm, 5 μm) reversed phase column with mobile phase consisting of acetonitrile and 2 mM ammonium acetate buffer (80:20, v/v) delivered in isocratic mode with constant flow rate of 0.7 mL/min. Detection of meropenem and rolipram was performed in positive mode using multiple reaction monitoring (MRM). The mass transitions 384.1→141.0 and 276.2→191.1 were used to quantify meropenem and rolipram, respectively. A fast and simple
Introduction
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Meropenem {chemically known as -[5-(dimethylcarbamoyl) pyrrolidin-2-yl] sulfanyl6- (1-hydroxyethyl)-4-methyl-7-oxo- 1-azabicyclo[3.2.0] hept-2-ene-2-carboxylic acid} is an ultra-broad spectrum injectable antibiotic used for treatment of wide variety of infections such as meningitis, urinary tract, febrile neutropenia, ▶ Fig. 1). pneumonia and cystic fibrosis [1–4] (● Meropenem was originally developed by Sumitomo Pharmaceuticals, but after approval by USFDA in 1996 it is currently available globally with brand names such as Merrem, Meronem, Zwipen, Mepem, Meropen and Neopenem etc. Meropenem is a high molecular weight drug (Mw 383.46) with poor and pH dependent aqueous solubility. It has 2 pKa values of 2.9 and 7.4 [5]. Meropenem is generally given by intravenous administration and supplied as white crystalline powder, to be dissolved in 5 % (w/v) potassium phosphate solution for intravenous administra-
solid phase extraction method was optimized for extraction of meropenem from rat plasma. The developed method was validated for selectivity, accuracy, precision, linearity, recovery, stability, matrix effect, dilution integrity as per regulatory guidelines. The developed method was selective with no interfering components at the retention time of meropenem and rolipram. The assay demonstrated acceptable linearity (R2 > 0.99) over a dynamic range of 0.19–201.40 μg/mL. The method exhibited excellent and acceptable precision and accuracy, and produced consistent recoveries. The method demonstrated excellent stability of meropenem in rat plasma under studied conditions investigated. Finally, the validated method was successfully applied to quantify meropenem levels in rat plasma of a dose escalation study.
tion. In humans, upon intravenous administration of 1 g of meropenem, Cmax levels up to 30 mg/ lit were observed. The elimination half life of meropenem was found to be 1 h with volume of distribution up to 21 lit. The Cmax and AUC of meropenem increase linearly with dose indicating the absence of saturation phenomenon [6]. It exhibits negligible plasma protein binding (up to 2 %) and penetrates well into most of body tissues including cerebrospinal fluid, heart and lung muscles, bile and peritoneal fluids. Even though the pharmacokinetics of meropenem in humans is extensively studied, the information related to disposition in laboratory animals such as rat is very limited. In addition, estimation of pharmacokinetics of meropenem in laboratory animals is essential during the safety assessment of toxicity of drug formulations in humans. Hence the objective of the present work was to develop a simple and efficient bioanalytical method for the quantification of meropenem in rat plasma.
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Development and Validation of Liquid Chromatography-Tandem Mass Spectrometric Method for Quantification of Meropenem in Rat Plasma and its Application in a Preclinical Dose Proportionality Study
322 Original Article
generation of charged ions in multiple reactions monitoring mode. The HPLC system (Perkin Elmer, Shelton, USA) consisting of PE 200 series autosampler, PE 200 series pump were used for injections of analyte solutions on to LC-MS/MS. The temperature of autosampler vial tray was maintained at 6 °C. The optimized mobile phase consisted of acetonitrile and 2 mM ammonium acetate (80:20, v/v) delivered at a flow rate of 0.7 mL/min. Upon 5 μL of injection on to a Kromasil 100 C18 reversed phase column (150 × 4.6 mm, 5 μ, Grace Davison, USA) the column eluents were monitored in mass spectrometer using transitions of 384.1→141.0 and 276.2→191.1 for meropenem and rolipram, respectively. The chromatograms were acquired using Analyst® software (Ver 1.4.1, Applied Biosystems, MDS SCIEX, Toronto, Canada).
Literature review revealed many high performance liquid chromatographic (HPLC) methods for the quantification of meropenem in pharmaceutical formulations and in biomatrices such as plasma, serum and bile [7–14]. The reported methods are time consuming, laborious and involved complex techniques such as online micro dialysis and column switching. In contrast to HPLC, modern liquid chromatography-tandem mass spectrometric (LC-MS/MS) methods offer ultra-fast analysis and involve minimal sample preparation. Only 2 LC-MS/MS methods have been reported for quantification of meropenem in cerebrospinal fluid and human plasma. The injection volumes used for quantification were upto 50 μL in published literature while 5 μL injection volume was used in present work [15, 16]. In the present work, with a lower limit of quantification, a simple and efficient LC-MS/MS method was developed and fully validated for quantification of meropenem for the first time in rat plasma. The method was applied for estimation meropenem in plasma samples of a dose escalation in rats.
Experimental
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Chemicals & reagents Meropenem (assay ≥ 87.6 %) working standard was supplied by Analytical Research Division of Ranbaxy Laboratories Limited, India. Rolipram (purity ≥ 98 %) was used as internal standard and procured from Sigma Aldrich, USA. HPLC grade acetonitrile and methanol were purchased from S.D. Fine Chemicals Ltd., Mumbai, India. Ammonium acetate and acetic acid were obtained from Sigma Aldrich, USA. Sodium bicarbonate and sodium citrate were purchased from Ranbaxy Fine Chemicals, New Delhi, India. Oasis HLB solid phase extraction cartridges (30 mg, 1 mL) were purchased from Waters corporation, Massachusetts, USA. Milli Q water (18.2 mΩ & TOC ≤ 50 ppb) from a Milli-Q® water purification system (Millipore SAS, Molshem, France) was used. All other chemicals and reagents used in the study were of ARgrade. The normal batches of Wistar Hannover rat plasma for method validation were obtained from in-house facility; while lipemic and hemolyzed batches were procured from Bioreclamation incorporation, Newyork, USA.
Instrumentation The quantification of meropenem in rat plasma was performed on API-4000 LC-MS/MS system (Applied Biosystems, MDS SCIEX, Toronto, Canada) coupled with electrospray ionization used for
Animals All the study protocols were approved by Institutional Animal Ethics Committee (IAEC, protocol: 68/08), Ranbaxy and were in accordance with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and OECD principles for Good Laboratory Practices (GLP). Healthy male Wistar Hannover rats with weight range of 130–250 g (4–7 weeks old) were obtained from Animal Breeding and Housing Facility, Ranbaxy Laboratories Limited, Gurgaon. After screening, the animals were placed in polycarbonate cage in groups of 4 and maintained in controlled environment conditions. Animals were acclimatized 7 days prior to commencement of work and they were provided autoclaved, standard pelleted laboratory animal diet, with free access to purified water.
Preparation of calibration curve and quality control samples The stock solution of meropenem was prepared by dissolving 20 mg of working standard in 1 mL of 0.5 % (w/v) sodium bicarbonate solution. Primary stock solution of meropenem for the preparation of calibration curve standard and quality control samples (QC) were prepared separately. Subsequent dilutions for calibration curve standard and quality control samples were prepared in 0.5 % (w/v) sodium bicarbonate solution. Spiking of aqueous dilutions from primary stock solution was done in plasma to get 8 point calibration curve (0.19–201.40 μg/mL) of meropenem. 8 non-zero of calibration curve standards were prepared by spiking 50 μL of each working stock into 950 μL of blank rat plasma to achieve final concentration of 0.19, 0.47, 1.18, 4.72, 18.88, 75.53, 151.05, 201.40 μg/mL. Quality control samples were prepared independently at 5 concentration level. These included the lower limit of quantification (LLOQQC, 0.19 μg/mL), within 3 times of lower limit of quantification (LQC, 0.48 μg/mL), in the middle (MQC, 80.48 μg/mL), near upper concentration (HQC, 160.96 μg/mL) and above upper concentration (DQC, 402.41 μg/mL). The aliquot of 75 μL of each CC & QC samples was stored below − 50 °C until analysis. The primary stock solution for internal standard (IS) was prepared in acetonitrile to obtained final concentration of 1.0 mg/mL. An IS working solution of 10.0 μg/mL was prepared by diluting primary stock with water/acetonitrile 50/50, v/v.
Sample preparation Solid phase extraction was used for extraction of drug from plasma. For sample preparation, 50 μL of plasma sample was mixed with 50 μL internal standard solution (Rolipram 10 μg/ mL) and 500 μL of 2 mM ammonium acetate buffer (pH adjusted
Chaursia BK et al. Method for Quantification of Meropenem … Drug Res 2014; 64: 321–329
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Fig. 1 Chemical structure of meropenem.
Original Article 323
Method development Electrospray (ESI) was selected as an ionization source in the present study. For tuning purpose, the standard solutions of meropenem and rolipram ( ≈ 250 ng/mL) were prepared in mixture of acetonitrile and MlliQ water (50:50, v/v) and were infused. Both analyte and internal standard were optimized in both positive and negative mode for obtaining maximum intensity. The parent and product ions for multiple reaction monitoring (MRM) were selected after scanning in Q1 MS, Q3 MS, product ion and precursor ion modes. After selection of mass transitions for MRM, various voltages such as declusterization potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) and collision activated dissociation (CAD) gas were optimized to produce maximum intensity. Other mass spectrometric parameters viz. nebulizer gas, heater gas, curtain gas, temperature, ion spray voltage were optimized by flow injection analysis (FIA). The separation of meropenem was achieved by liquid chromatographic technique by optimizing combination of water, various buffer solutions, acetonitrile and methanol. The peak properties of meropenem were optimized by selection of suitable reversed phase column. Other chromatographic parameters like flow rate and injection volume were optimized for better sensitivity, good resolution, as well as shorter run time for analyte and IS. Several internal standards were tried; however chromatography, consistent response and reproducibility were obtained with rolipram. Therefore rolipram was selected as internal standard. Following optimization of mass spectrometer condition, each step in processing, such as sample preparation, chromatographic separation of interfering components from analyte peak, extraction efficiency were optimized for developing a sensitive and reproducible bioanalytical method. Various sample clean up techniques such as protein precipitation using methanol and acetonitrile, liquid-liquid extraction using various organic solvents such as ethyl acetate, tertiary methyl butyl ether, dichloromethane, n-hexane alone and in combination were evaluated for maximizing extraction efficiency. Finally solid phase extraction using various sorbents and various combinations of washing and eluting solvents was performed for enhanced sample clean-up and extraction efficiency.
Method validation The developed bioanalytical method for quantification of meropenem in rat plasma was validated with respect to various parameters such as selectivity, linearity and range, sensitivity, accuracy and precision, carry over, recovery, matrix effect, stability of meropenem in rat plasma under various conditions, dilution integrity and reinjection reproducibility as per USFDA guidelines for bioanalytical method validation [17].
Application of bioanalytical method to a dose escalation study in rat The dose escalation study in rat was performed by administering meropenem at various dose levels such as 500, 1 000 and 2 000 mg/kg by intravenous route through caudal vein. After a predefined inclusion and exclusion criteria, total of 63 animals were selected and divided into 3 groups of 21 for each dose level. The formulations were prepared by dissolving meropenem in dextrose solution (10 %, w/v) to achieve 50, 100 and 200 mg/mL solutions and were used immediately to dose 500, 1 000 and 2 000 mg/kg respectively. The dose volume was 10 mL/kg of the body weight of animal. The administration was done at the rate of 2 mL/min using a calibrated infusion pump (Harvard Apparatus, Mumbai, India). Approximately, 250 μL of blood samples were collected from retro-orbital plexus at 0.083, 0.25, 0.5, 1.0, 1.5, 2.0, 4.0 h in an Eppendorf tube containing 10 μL of sodium citrate solution (20 %, w/v). 3 rats were used for a single time point and the blood samples were immediately placed on wet ice. The collected blood samples were centrifuged (at 10 000 rpm, 4 °C for 5 min) within 30 min of collection to separate plasma and stored immediately below − 50 °C until analysis. The concentration-time data was analyzed using validated WinNonlin Professional software (Version 5.0.1, Pharsight Corporation, Cary, North Carolina) and the pharmacokinetic parameters such as Cmax, T1/2, AUC0–t and AUC0–∞ were estimated by non-compartmental approach at each dose level. The Cmax is calculated as the highest concentration observed in the profile. The elimination rate constant (λz) was obtained from the slope of linear regression line of log transformed concentration vs. time profile. The elimination half life is calculated from λz using the expression 0.693/λz. The area under the curve till last measurable concentration (AUC0–t) was calculated from log-linear trapezoidal rule. The area under curve extrapolated to infinity (AUC0–∞) was calculated from the expression AUC0–t + Ct/λz, where Ct is the last measurable concentration.
Results and Discussion
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Method development Depending on the polarity and molecular weight of meropenem and rolipram, ESI was selected as an ionization source in the present study. Both the molecules showed maximum intensity in Q1MS in positive ion mode in comparison to negative mode and it was selected for further optimization. In Q1MS scan mode the, most abundant protonated ion [M + H] + for meropenem ▶ Fig. 2), and rolipram (276.2) were selected. In the (m/z 384.1) (● product ion mode, the values of CE and CAD were varied and optimized for selection of most abundant product ion for mero▶ Fig. 3) and rolipram (191.1). Moreover, the penem (141.0) (● parent ions for selected product ions were confirmed by scanning in precursor mode for both meropenem and rolipram. After the selection of parent to product ion transition, various MS parameters like DP, EP and CXP were optimized for meropenem and rolipram in MRM mode. The optimized values of MS param▶ Table 1. After eters for meropenem and rolipram are given in ● the optimization of MS parameters, chromatographic optimization was performed aiming at better sensitivity and superior peak properties. Experiments carried out with combination of water and methanol (50:50, %v/v) using a C18 reversed phased column showed peak tailing for meropenem. Replacing methanol
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to 4.8 with glacial acetic acid) and vortexed for homogeneous mixing. The content was transferred to Oasis HLB extraction cartridge preconditioned with 1 mL methanol and 1 mL of 2 mM ammonium acetate buffer (pH 4.8) at a constant pressure of 10–15 psi. The loaded cartridge was washed with 1 mL of 2 mM ammonium acetate buffer (pH 4.8) and 1 mL of MilliQ water to remove matrix components. Finally, the elution was carried out into a clean glass tube twice with 700 μL of elution solution (acetonitrile/2 mM ammonium acetate buffer (pH 4.8, 50:50, v/v). The samples were vortexed for homogeneity and 5 μL was injected on to LC-MS/MS for analysis.
324 Original Article
Fig. 3 Product ion mass spectrum of meropenem for selection of product ion.
with acetonitrile resulted in improved peak properties for both meropenem and rolipram but resulted in poor retention. Further optimization of peak properties was carried out by incorporation of buffering agents such as ammonium acetate (2 mM) into the mobile phase. Among all combinations of acetonitrile
and 2 mM ammonium acetate, 80:20 ( %v/v) showed better peak parameters and improved retention for both meropenem and rolipram. Further optimization of retention was carried out by optimizing the flow rate to 700 μL/min and injection volume of 5 μL was selected based on the signal to noise ratio (S/N) in
Chaursia BK et al. Method for Quantification of Meropenem … Drug Res 2014; 64: 321–329
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Fig. 2 Q1MS spectrum of meropenem for selection of parent ion.
Original Article 325
Table 1 Optimized MS parameters for meropenem and rolipram. MS Parameter
Meropenem
Rolipram
Transition (m/z) DP (Volt) EP (Volt) CE (Volt) CXP (Volt) Nebulizer gas (Psi) Heater gas (Psi) Temperature ( °C) Ion spray voltage (Volt)
384.1/141.0 85 10 22 8
276.2/191.1 80 10 24 8 50 50 500 5 500
Method validation Selectivity Chromatograms of blank samples revealed no interfering peaks eluting at the retention time of meropenem or rolipram. Lack of response from endogenous impurities resulting from biomatrix indicated the selectivity of the method for quantification of meropenem from rat plasma. Comparison of chromatograms of blank sample, zero sample and LLOQ sample confirmed the ▶ Fig. 4–6). In addition, analysis of selectivity of the method (● blank and LLOQ samples from hemolyzed and lipemic plasma resulted in no significant effect of endogenous impurities on the quantification of meropenem, indicating the applicability of method for various types of plasma samples.
Linearity The linear regression analysis indicated the linear relationship between meropenem concentration and area ratio (meropenem to rolipram) over a dynamic range of 0.19–201.4 μg/mL. The coefficient of linear regression (R2) values was greater than 0.99 in all analytical batches indicating the linearity of proposed method. Weighting factor 1/x2 was chosen based on the concentration-response relationship. The best fit linear regression equation estimated from linear regression analysis was: peak area ratio (Analyte/IS) = 0.0174 ± 0.0021 × Concentration of meropenem (μg/mL) + 0.0000943 ± 0.0001. The calculated intercept was significantly low indicating the accuracy of the method and insignificant contribution of matrix components in the quantification of meropenem.
Precision and accuracy The obtained results for %CV and %nominal represent the precision and accuracy of the proposed method as the values were in accordance with the regulatory acceptance criteria. The results ▶ Table 2a, b. The overall of precision and accuracy are shown in ● within precision and accuracy ranged from 1.93–11.29 % and 93.80–104.24 %, respectively. The intra-day precision and accuracy values ranged from 2.46–9.52 % and 93.80–103.28 %, respectively. In addition, the inter-day precision accuracy ranged from 2.90–8.20 % and 95.91–101.73 %, respectively. The extended
Fig. 4 Representative chromatogram for blank rat plasma.
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processed LLOQ samples. Based on all the above trials, the optimized mobile phase combination consisting of acetonitrile and 2 mM ammonium acetate (80:20, %v/v) and the retention times of meropenem and rolipram in this combination were 1.5 and 2.5 min, respectively over a run time of 3 min. For sample preparation, a single step precipitation with methanol or acetonitrile resulted in insufficient sample clean up and high protein load resulting in increasing back pressure of the column. Liquid liquid extraction with ethyl acetate, tertiary methyl butyl ether, dichloromethane, n-hexane alone and in various combinations showed poor extraction efficiency for meropenem. Acidification of plasma showed marginal increase in recovery for meropenem. In order to increase extraction efficiencies, produce a cleaner chromatogram, overcome the problems of high back pressure and improper peak shape, solid phase extraction was evaluated for the extraction of meropenem from plasma. Solid phase extraction using Oasis HLB cartridges provided better recovery as compared to other cartridges. As meropenem is weakly acidic (pKa 2.9, 7.4), the conditioning and washing steps during solid phase extraction were optimized with 2 mM ammonium acetate (pH, 4.8) for better retention of meropenem on the solid phase sorbent. Finally, the elution of meropenem was carried out using a combination of acetonitrile and 2 mM ammonium acetate (pH, 4.8) (50:50, v/v) for better recoveries from rat plasma.
326 Original Article
Fig. 6 Representative chromatogram for lower limit of quantification.
batch precision and accuracy values ranged from 4.97–7.30 % and 91.09–99.64 %, respectively indicating the precision and accuracy of the proposed method over an analytical batch consisting of 120 samples.
Carryover effect The method demonstrated no carryover for meropenem and rolipram as there was no observed peak area for meropenem and rolipram in any of the 2 blank samples injected after the upper limit of quantification samples.
Sensitivity The plasma standards prepared at LLOQ level were analyzed in 5 replicates in 3 analytical batches. The comparison of chromato▶ Fig. 6) with blank sample (● ▶ Fig. 4) indicate gram of LLOQ (● the sensitivity of the method as the obtained S/N ratio was significantly higher than 5. The between batch precision and accuracy values at LLOQ in 3 analytical batches were 5.00 % and 105.26 %, respectively demonstrating the sensitivity of the method for quantification of meropenem.
Matrix effect The method demonstrated insignificant matrix effect from endogenous impurities in the quantification of meropenem. The matrix factor values obtained during the method development for meropenem and rolipram were 0.98 ± 0.0298 and 0.98 ± 0.0666, respectively indicating the negligible contribution of matrix components in the quantification of meropenem. Moreover, the value of IS normalized matrix factor was 1.009 ± 0.040 indicating the similar matrix effect for meropenem and rolipram. The obtained within batch precision and accuracy values during validation from 12 sets of LQC and HQC ranged from 1.16–4.46 % and 95.68–104.34 %.
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Fig. 5 Representative chromatogram for blank rat plasma spiked with rolipram.
Original Article 327
QC
LLOQ QC
LQC
MQC
HQC
Nominal Concentration (μg/mL) Batch-1 Mean SD CV %nominal N Batch-2 Mean SD CV %nominal N Batch-3 Mean SD CV %nominal N Extended batch Mean SD CV %nominal N
0.19
0.48
80.48
160.96
0.19 0.02 11.29 101.05 5
0.46 0.01 1.93 96.67 5
82.34 5.27 6.40 102.31 5
156.59 5.08 3.24 97.29 5
0.19 0.01 3.72 100.00 5
0.48 0.01 2.29 98.58 5
83.90 4.53 5.40 104.24 5
155.55 6.21 3.99 96.64 5
0.19 0.02 9.52 98.95 5
0.47 0.02 3.82 97.50 5
79.38 4.76 5.99 98.63 5
150.97 3.71 2.46 93.80 5
0.44 0.02 5.41 91.09 40
80.19 5.86 7.30 99.64 40
153.33 7.62 4.97 95.26 40
LLOQ QC
LQC
MQC
HQC
Nominal Concentration (μg/mL) Within Batch Mean SD CV %nominal N Intra-day (Batch 1&2) Mean SD CV %nominal N Intra-day (Batch 3) Mean SD CV %nominal N Inter-day Mean SD CV %nominal N
The absolute recoveries of meropenem and rolipram were calculated by analyzing the 5 replicates of LQC, MQC and HQC and comparing against freshly prepared aqueous samples. The proposed method produced consistent absolute recoveries at all studied levels such as LQC, MQC and HQC, respectively. The overall recovery for meropenem and rolipram were calculated to be 38.39 ± 5.82 and 121.11 ± 8.54, and %CV were 15.17 & 7.05 respectively. The precision at LQC, MQC and HQC levels were 3.80, 6.01 and 5.06, respectively. The consistent recoveries indicate that the developed method was precise and accurate for quantification of meropenem in rat plasma.
Stability ▶ Table 3. The results The result of stability exercises is given in ● demonstrate that meropenem is stable under various storage and processing conditions. No significant degradation of meropenem was observed upon 3 freeze thaw cycles as the mean stability ranged from 92.92–97.97 % and 93.33–97.83 %, respectively for 1 and 3 freeze thaw cycles. The LQC and HQC samples stored at room temperature over wet ice demonstrated the stability of meropenem as the mean accuracy ranged from 102.34–103.33 % after 2 h. Long term stability study showed that meropenem is stable in rat plasma up to 39 days when stored below − 50 °C as there was no significant change in response when compared with 0 h samples. The mean accuracy values for meropenem up to 39 days ranged from 99.17–100.76 %. The processed LQC and HQC samples were stable in autosampler conditions up to 24 h as the mean accuracy values for meropenem and rolipram ranged from 93.35–94.58 % and 98.21–104.95 %, respectively.
Dilution integrity
Table 2b Precision and accuracy data for meropenem in rat plasma. QC
Recovery
0.19
0.48
80.48
160.96
0.19 0.01 3.72 100.00 5
0.48 0.01 2.29 99.58 5
83.90 4.53 5.40 104.24 5
155.55 6.21 3.99 96.64 5
0.19 0.02 7.98 100.53 10
0.47 0.01 2.54 98.13 10
83.12 4.70 5.66 103.28 10
156.07 5.37 3.44 96.96 10
0.19 0.02 9.52 98.95 5
0.47 0.02 3.82 97.50 5
79.38 4.76 5.99 98.63 5
150.97 3.71 2.46 93.80 5
0.19 0.02 8.20 100.00 15
0.47 0.01 2.90 97.92 15
81.87 4.90 5.99 101.73 15
154.37 5.35 3.47 95.91 15
The developed method demonstrated over curve dilution integrity for 10, 20 and 40-folds dilution with rat plasma. The within batch accuracy values for 10, 20 and 40-folds dilution were 107.34, 95.72 and 103.60 %, respectively.
Reinjection reproducibility The reinjection reproducibility of the method was evaluated to demonstrate the integrity of meropenem upon piercing the sample closure. The results demonstrated reinjection reproducibility of meropenem up to 3 injections as the %initial with respect to first injection ranged from 92.31–102.28 % at LQC and HQC.
Application of bioanalytical method to a dose escalating study Plasma concentrations of meropenem in rat declined monoexponentially and were below the limit of quantitation by 4 h post dose. The mean plasma concentration vs. time profiles of mero▶ Fig. 7, 8. The penem at all studied dose levels are overlaid in ● mean pharmacokinetic parameters of the parent compound are ▶ Table 4. The exposures (AUC outlined in ● 0–t) increased proportional to the dose (R2 > 0.95) and were found to be 232.92, 314.69, 940.10 h.μg/mL respectively following 500, 1 000 and 2 000 mg/kg doses. AUC0–t at all dose levels were more than 95 % of AUC0–∞ indicating that the samples were collected long enough to determine total exposure from the doses administered. Like other penams, meropenem is also cleared very rapidly in the rat with short half life and small volume of distribution (Vd).
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Table 2a Precision and accuracy data for meropenem in rat plasma (Individual batches).
328 Original Article
QC Sample
Stability
Calculated conc. (μg/mL)*
LQC HQC
Freeze-thaw (1 cycles)
LQC HQC
Freeze-thaw (3 cycles)
LQC HQC
Bench top (2 h in wet ice)
LQC HQC
Autosampler (24 h)
LQC HQC
Long term (39 days)
0.45 ± 0.01 157.69 ± 2.36 0.45 ± 0.01 157.47 ± 1.44 0.50 ± 0.02 164.72 ± 1.05 0.45 ± 0.01 150.26 ± 2.72 0.48 ± 0.02 162.18 ± 0.48
% CV
% nominal
2.01 1.50 2.91 0.91 2.01 1.50 1.97 1.81 3.52 2.95
92.92 97.97 93.33 97.83 103.33 102.34 94.58 93.35 99.17 100.76
Table 3 Stability data for meropenem in rat plasma.
*each value is average of 5 determinations and results are expressed as Mean ± SD
Conclusion In the present work, a new, simple LC-MS/MS method was developed and validated for quantitative determination of meropenem from rat plasma. The method was fully validated for various method validation parameters as per regulatory guidelines. Simple and fast solid phase extraction resulted in consistent and reproducible recoveries of meropenem over the dynamic range. The method was found to be accurate, precise with excellent stability of meropenem under various processing and storage conditions. Finally, the method was successfully employed for determining the dose proportionality of meropenem upon intravenous administration in rats. Fig. 7 Mean plasma concentration vs. time profile of meropenem upon intravenous administration at all studied dose levels.
Conflict of Interest
▼
Authors of this publication do not have any Conflict of Interest.
References
Fig. 8 Semi log plot of mean plasma concentration vs. time profile of meropenem upon intravenous administration at all studied dose levels.
Table 4 Mean pharmacokinetic parameters of meropenem in rats upon intravenous administration. Dose
Tmax
Cmax
AUC0–t
AUC0–∞
Cl
(mg/kg)
(h)
(μg/mL)
(h.μg/mL)
(h.μg/mL)
(mL/h/kg) (mL/kg)
500 1 000 2 000
0.083 836.24 232.92 0.083 1 127.47 314.69 0.083 3 363.33 940.10
233.24 315.10 940.46
2 143.67 3 173.55 2 126.60
Vd 287.61 434.08 290.33
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