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A rapid, sensitive high performance liquid chromatographic method for the determination of meropenem in pharmaceutical dosage form, human serum and urine Yalcin O È zkan, 1 I Ç lkay Ku Èc Ëu È kgu È zel, 2 Sibel A. O È zkan 3 and Hassan Y. Aboul-Enein 4 * 1 Gu È lhane Military Medical Academy, Department of Pharmaceutical Sciences, 06018 Ankara, Turkey 2 Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Haydarpasa, Istanbul, Turkey 3 Ankara University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 Ankara, Turkey 4 Pharmaceutical Analysis Laboratory, Department of Biological & Medical Research, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia Received 18 July 2000; accepted 18 September 2000 ABSTRACT: A new, simple, precise and rapid high performance liquid chromatographic method was developed for the determination of meropenem in human serum, urine and pharmaceutical dosage forms. Chromatography was carried out on an LC 18 column using a mixture of 15 mM KH 2 PO 4 :acetonitrile:methanol (84:12:4; v/v/v), adjusted to pH 2.8 with H 3 PO 4 . The proposed method was conducted using a reversed-phase technique, UV monitoring at 307.6 nm and cefepime as an internal standard. The retention times were 5.98 and 7.47 min for cefepime and meropenem, respectively. The detector response was linear over the concentration range of 50–10,000 ng/mL. The detection limit of the procedure was found to be 22 ng/mL. The detection limit for meropenem in human plasma was 108.4 ng/mL and the corresponding value in human urine was 179.3 ng/mL. No interference from endogenous substances in human serum, urine and pharmaceutical preparation was observed. The proposed method is sufficiently sensitive for determination of the concentrations of meropenem and may have clinical application for its monitoring in patients receiving the drug. Copyright # 2001 John Wiley & Sons, Ltd. INTRODUCTION Meropenem (Fig. 1) is an active, broad spectrum carbapenem antibiotic. Its in vitro activity is similar to that of imipenem, with activity against some imipenem- resistant Pseudomonas aeruginosa, but less activity against Gram-positive cocci. Clinical experience with meropenem is limited (Mandell and Petri, 1996; Gold and Moellering 1996). To date, few high performance liquid chromatographic (HPLC) methods have been described for determining meropenem in biological samples (Al-Meshal et al., 1995; Ip et al., 1998) and intravenous solutions (Spell and Stewart, 1999), both of which are sensitive but quite complicated and time-consuming. It has also been determined by a carbon dioxide adduct using proton NMR and flow-injection quadropole mass spectrometry (Almarsson et al., 1998). In the modern analytical laboratory, there is always a need for faster, simpler, cheaper and better performance analytical methods. For this reason, we developed a method that is simpler, faster and easier than the previously published HPLC procedures and sensitive enough to determine meropenem plasma and urine concentrations. This paper describes the experimental conditions for the determination of meropenem in human serum, urine and pharmaceutical dosage forms by reversed-phase (RP)-HPLC method. EXPERIMENTAL Apparatus. The HPLC system used consisted of: a high-pressure, constant-volume pump (Waters model 510; Waters Assoc., Milford, MA, USA), UV detector (Waters, model 481), an autosampler (model 717 plus), an LC 18 column (150 4.6 mm; 5 mm particle size; Waters Assoc.). Chemicals and reagents. Meropenem was kindly provided by Figure 1. Chemical structure of meropenem. BIOMEDICAL CHROMATOGRAPHY Biomed. Chromatogr. 15: 263–266 (2001) DOI: 10.1002/bmc.68 *Correspondence to: H. Y. Aboul-Enein, Pharmaceutical Analysis Laboratory, Department of Biological and Medical Research (MBC- 03). King Faisal Specialist Hospital and Research Centre, PO Box 3354, Riyadh 11211, Saudi Arabia. E-mail: [email protected] Copyright 2001 John Wiley & Sons, Ltd. ORIGINAL RESEARCH

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Page 1: A rapid, sensitive high performance liquid chromatographic method for the determination of meropenem in pharmaceutical dosage form, human serum and urine

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Received 18 July 2000; accepted 18 September 2000

ABSTRACT: A new, simple, precise and rapid high performance liquid chromatographic method was developed for thedetermination of meropenem in human serum, urine and pharmaceutical dosage forms. Chromatography was carried out on an LC18

column using a mixture of 15 mM KH2PO4:acetonitrile:methanol (84:12:4; v/v/v), adjusted to pH 2.8 with H3PO4. The proposedmethod was conducted using a reversed-phase technique, UV monitoring at 307.6 nm and cefepime as an internal standard. Theretention times were 5.98 and 7.47 min for cefepime and meropenem, respectively. The detector response was linear over theconcentration range of 50–10,000 ng/mL. The detection limit of the procedure was found to be 22 ng/mL. The detection limit formeropenem in human plasma was 108.4 ng/mL and the corresponding value in human urine was 179.3 ng/mL. No interference fromendogenous substances in human serum, urine and pharmaceutical preparation was observed. The proposed method is sufficientlysensitive for determination of the concentrations of meropenem and may have clinical application for its monitoring in patientsreceiving the drug. Copyright � 2001 John Wiley & Sons, Ltd.

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Meropenem (Fig. 1) is an active, broad spectrumcarbapenem antibiotic. Its in vitro activity is similar tothat of imipenem, with activity against some imipenem-resistant Pseudomonas aeruginosa, but less activityagainst Gram-positive cocci. Clinical experience withmeropenem is limited (Mandell and Petri, 1996; Gold andMoellering 1996).

To date, few high performance liquid chromatographic(HPLC) methods have been described for determiningmeropenem in biological samples (Al-Meshal et al.,1995; Ip et al., 1998) and intravenous solutions (Spell andStewart, 1999), both of which are sensitive but quitecomplicated and time-consuming. It has also beendetermined by a carbon dioxide adduct using protonNMR and flow-injection quadropole mass spectrometry(Almarsson et al., 1998).

In the modern analytical laboratory, there is always aneed for faster, simpler, cheaper and better performanceanalytical methods. For this reason, we developed amethod that is simpler, faster and easier than the

previously published HPLC procedures and sensitiveenough to determine meropenem plasma and urineconcentrations. This paper describes the experimentalconditions for the determination of meropenem in humanserum, urine and pharmaceutical dosage forms byreversed-phase (RP)-HPLC method.

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���������$ The HPLC system used consisted of: a high-pressure,constant-volume pump (Waters model 510; Waters Assoc.,Milford, MA, USA), UV detector (Waters, model 481), anautosampler (model 717 plus), an LC18 column (150 � 4.6 mm;5 �m particle size; Waters Assoc.).

0 ������ �� �����$ Meropenem was kindly provided by

Figure 1. Chemical structure of meropenem.

BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 15: 263–266 (2001)DOI: 10.1002/bmc.68

*Correspondence to: H. Y. Aboul-Enein, Pharmaceutical AnalysisLaboratory, Department of Biological and Medical Research (MBC-03). King Faisal Specialist Hospital and Research Centre, PO Box3354, Riyadh 11211, Saudi Arabia.E-mail: [email protected]

Copyright 2001 John Wiley & Sons, Ltd.

ORIGINAL RESEARCH

Page 2: A rapid, sensitive high performance liquid chromatographic method for the determination of meropenem in pharmaceutical dosage form, human serum and urine

Zeneca Pharmaceuticals Inc. (Istanbul, Turkey). The internalstandard, cefepime HCl (cefepime), was received from Bristol-Myers-Squibb Pharmaceuticals Inc. (Istanbul, Turkey). Acetonitrileand methanol were of HPLC grade, purchased from Merck(Darmstadt, Germany). All other chemicals were commercialanalytical grade. Doubly distilled water was used for preparingsolutions.

0 ���������� �� ��������$ The proposed method wasconducted using a reversed-phase technique, UV monitoring at307.6 nm (AUFS 0.01) and cefepime as an internal standard. Amixture of 15 mM KH2PO4:acetonitrile:methanol (84:12:4; v/v/v),adjusted to pH 2.8 with H3PO4 was used as a mobile phase. Themobile phase was prepared daily, filtered, sonicated before use,and delivered at a flow rate of 1.0 mL/min.

"������ ����� �������$ Internal standard solution wasprepared by dissolving 10 mg cefepime HCl in 10 mL distilledwater. Accurately weighed 10 mg of standard meropenem wastaken in a 10 mL volumetric flask. A 10 mL aliquol of distilledwater was added and kept in an ultrasonic bath for 10 min.

Standard solutions for HPLC were prepared with mobile phase byvarying the concentration of meropenem in the range of 50–10,000 ng/mL and maintaining the concentration of cefepime(internal standard) at a constant level of 3000 ng/mL. Peak-arearatios were used to prepare calibration graphs and to calculate theconcentrations.

"���� ���������$ No sample preparation for the intravenousvials was required other than dissolving in distilled water anddilution with the mobile phase.

Serum and urine samples, obtained from healthy individuals(after obtaining their written consent), were stored frozen untilassay. After gentle thawing, 500 �L aliquots of serum were fortifiedwith an appropiriate amounts of meropenem (dissolved in distilledwater), maintaining the concentration of cefepime at a constantlevel. The tubes were vortexed for 30 s and then centrifuged for10 min at 4000g. The supernatant was discarded carefully. Serumsamples including various concentration of meropenem andconstant amount of cefepime were injected into the HPLC column.The determination of meropenem in urine required a solid phaseextraction with Sep-Pak NH2 cartridges. The urine sample (2 mL)was transferred into a tube containing appropriate amount ofmeropenem and internal standard. The tube was agitated vigorouslyon a vortex for 30 s and after that the mixture was applied to a Sep-Pak NH2 cartridge. The catridge was washed 2 mL distilled waterand any retained meropenem together with internal standard waseluted using 1 mL of 0.1 N HCl. Finally the eluted analytes wasdirectly injected into the HPLC column. Calibration curves werealso obtained from human serum and urine.

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A reversed-phase HPLC procedure was proposed as asuitable method for the determination of meropenem inhuman serum, urine and pharmaceutical preparations.The chromatographic conditions were adjusted in orderto provide a versatile HPLC procedure capable ofseparating meropenem and cefepime (internal standard).A mixture of 15 mM KH2PO4, acetonitrile and methanol(84:12:4; v/v/v) adjusted to pH 2.8 with H3PO4 at a flowrate of 1 mL/min was found to be an appropriate mobilephase allowing adequate separation of active substanceand the internal standard. Figure 2 shows a typicalchromatogram obtained from the analysis of a standardmeropenem solution using the proposed method. As

Figure 2. Typical chromatogram of a standard solution ofmeropenem (1500 ng/mL) and cefepime (IS) (3000 ng/mL) inthe mobile phase.

Table 1. Characteristics of meropenem calibration plot obtained from standard solutions, serum and urine samples

MediumConcentrationrange (ng/mL) Slope Intercept

Correlcoefficient

Standard errorof slope

Standard errorof intercept

Detection limit(ng/mL)

Mobile phase 50–10,000(n = 8)

9.44 � 10�4 �8.07 � 10�3 0.999 3.17 � 10�6 0.0013 22.12

Serum 500–10,000(n = 6)

5.43 � 10�4 �0.105 0.999 5.77 � 10�6 0.028 108.4

Urine 750–10,000(n = 6)

8.82 � 10�4 �4.44 � 10�2 0.998 �2.68 � 10�5 0.014 179.3

Copyright 2001 John Wiley & Sons, Ltd. Biomed. Chromatogr. 15: 263–266 (2001)

264 ORIGINAL RESEARCH Y. Ozkan et al.

Page 3: A rapid, sensitive high performance liquid chromatographic method for the determination of meropenem in pharmaceutical dosage form, human serum and urine

shown in Fig 2, meropenem and cefepime were eluted,forming well-shaped, symmetrical peaks, well separatedfrom the solvent front. The retantion times were 5.73 minfor cefepime and 7.13 min for meropenem.

Table 1 represents a calibration plot for the peak-arearatio of varying amounts of meropenem (50–10,000 ng/mL) to a constant amount of cefepime (3000 ng/mL). Theinjection volume was 50 �L. Calibration characteristicswere given in Table 1. The limit of detection of the

procedure was shown in Table 1, which was calculated asthe blank response plus three times the blank standarddeviation devided by the slope of calibration curve.

Table 2 represents the results obtained for within- andbetween-day variability studies of meropenem samples.The within-day precision for the studied concentrations(250 and 2500 ng/mL) showed relative standard devia-tions (RSD%) 0.52 and 0.14, respectively. The evaluatedbetween-day precision obtained over a 2 week periodvaried from 0.75 to 1.65%. These results show the

Table 2. Within-day and between-day precision of meropenem standards

Theoretical concentration Within-day measured concentrationa (ng/mL) Between-day measured concentrationb (ng/ml)

(ng/mL) Mean RSD % Mean RSD %

250 251.2 0.522 251.9 0.752500 2502 0.136 2513.4 1.65

a Mean values represent five different meropenem standards for each concentration.b Between-day reproducibility was determined from five different runs over a 2 week period.

Figure 3. Chromatogram of blank serum (a) and serum spikedwith 1500 ng/mL of meropenem and 3000 ng/mL of cefepime(IS) (b).

Figure 4. Chromatogram of blank urine (a) and urine spikedwith 1500 ng/mL of meropenem and 3000 ng/mL of cefepime(IS) (b).

Table 3. Results obtained for meropenem analysis from biological samples

SampleMeropenem added

(ng/mL) nMeropenem found

(ng/mL) RSD (%)Average recovery

(%)

Serum 1500 4 1480.3 1.12 98.73000 4 2944.0 1.17 98.1

Urine 1500 4 1440.8 1.25 96.073000 4 2896.2 1.58 96.54

Copyright 2001 John Wiley & Sons, Ltd. Biomed. Chromatogr. 15: 263–266 (2001)

Determination of meropenem ORIGINAL RESEARCH 265

Page 4: A rapid, sensitive high performance liquid chromatographic method for the determination of meropenem in pharmaceutical dosage form, human serum and urine

accuracy and precision of the assay. Thus, it wasconcluded that there was no significant difference forthe assay which was tested within-day and between-day.

Figure 3(a) shows a typical chromatogram of anextract of fresh blank plasma, and Fig. 3(b) shows achromatogram obtained when the method was applied tospiked plasma containing 3000 ng/mL of meropenem and3000 ng/mL of the internal standard. The internalstandard and meropenem gave well-separated sharpsymmetrical peaks with retention times of 5.85 and6.97 min, respectively. There are no extraneous peaks inchromatograms obtained for serum samples. The calibra-tion graph obtained by spiking a fixed amount ofcefepime (3000 ng) and meropenem in the range 500–10,000 ng into 1 mL of human plasma was linear. Table 1summarizes the characteristics of the calibration graphobtained from serum samples. The detection limit ofmeropenem in human serum was 108.4 ng/mL (Table 1),which was calculated as the blank response plus threetimes the blank standard deviation devided by the slopeof calibration curve.

This RP-HPLC method gives reproducible results,easy to perform and is sensitive enough for thedetermination of meropenem in human plasma (Table 3).

Typical chromatograms of a human urine blank and asample spiked with meropenem are shown in Fig. 4(a) and(b), respectively. The separation of meropenem andcefepime from endogenous substances was satisfactory.The retention times of cefepime and meropenem wereobtained at 5.85 and 7.07 min, respectively. The ratios ofmeropenem peak heights to those of the internal standard

were linearly related to meropenem concentration inhuman urine over range 750–10,000 ng/mL. Table 3 showsmeropenem levels in urine spiked with meropenem. Theresults indicate that proposed HPLC method is suitable formonitoring the levels of meropenem in human urine.

The applicability of the method was also tested byanalysing meropenem in pharmaceutical dosage forms.Figure 5 shows a typical chromatogram obtained for theanalysis of meropenem in intravenous vials. The results ofthe analysis of meropenem intravenous vials (Table 4)indicate that theproposedassaycanbeusedforquantitationand routine quality control analysis of meropenem indosageforms.Itcanbeconcludedfromtheseresults that theproposed method is sufficiently accurate and precise to beapplied to the analysis of meropenem in pharmaceuticaldosage forms, human serum and urine within a shortanalysis time (�8 min). The described HPLC method issimple, requiring minimum sample handling and time. It issensitive enough for the determination of low amounts ofmeropenem in human serum and urine. A major advantageof this new method, which involves Sep-Pak NH2 solidphase extraction for urine samples and no extraction steprequired for serum samples and pharmaceutical formula-tions, is that it allows a rapid, simple and sensitivedetermination without extensive and time-consumingclean-up procedures, which may influence results.

No interferences from endogenous substances wereobserved in any of the biological samples. The proposedmethod should be useful for the therapeutic monitoring oflevels of meropenem in biological samples and may haveclinical application for patients receiving the drug.

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Almarsson O, Kaufman MJ, Stong JD, Wu Y, Mayr SM, Petrich MAand Williams JM. Journal of Pharmaceutical Science 1998; 87: 663.

Al-Meshal MA, Ramadan MA, Lotfi KM and Shibl AM. Journal ofClinical Pharmacology Therapeutics 1995; 20: 159.

Gold HS and Moellering RC Jr. New England Journal of Medicine1996; 335: 1445.

Mandell GL and Petri WA (eds). Goodman & Gillman’s ThePharmacological Basis of Therapeutics, 9th edn. McGraw-Hill:New York, 1996, 1057.

Ip M, Au C, Cheung SW, Chan CY and Cheng AFB. Journal ofAntimicrobial Chemotherapy 1998; 42: 121.

Spell JC and Stewart JT. Journal of Liquid Chromatography andRelated Technologies 1999; 22: 2225.

Figure 5. Chromatogram obtained from a pharmaceuticaldosage form containing 3000 ng/mL meropenem and 3000 ng/mL cefepime (IS).

Table 4. Results of the determination of meropenem inpharmaceutical formulations

Label claim(g)

Mean of amounta

found (g) RSD% 95 % CI

0.5 0.499 0.54 3.33 � 10�3

1.0 0.996 1.41 2.99 � 10�2

a Each value is the mean of five experiments.

Copyright 2001 John Wiley & Sons, Ltd. Biomed. Chromatogr. 15: 263–266 (2001)

266 ORIGINAL RESEARCH Y. Ozkan et al.