American Journal of Analytical Chemistry
Vol.06 No.03(2015), Article ID:53816,6 pages

Determination of Antibiotic Drug Cefdinir in Human Plasma Using Liquid Chromatography Tandem Mass Spectroscopy

Mohammad A. Al Bayyari1, Raed S. Abu Ajjour2

1College of Science and Health Professions, King Saud Bin Abdulaziz University for Health Sciences, National Guard Health Affairs, Jeddah, KSA

2Bioanalytical Laboratory, International Pharmaceutical Research Center, Amman, Jordan


Copyright © 2015 by authors and Scientific Research Publishing Inc.

This work is licensed under the Creative Commons Attribution International License (CC BY).

Received 27 December 2014; accepted 2 February 2015; published 5 February 2015


A highly sensitive, accurate and rapid analytical method based on reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (RP-LC-ESI-MS/MS) has been developed and validated for the determination of cefdinir in human plasma using cephalexin as an internal standard (IS). The method was validated over a linear range of 10 - 1200 ng/ml. After addition of IS, analytes were extracted from the plasma samples by precipitation extraction using mixture of 0.1% formic acid solution in acetonitrile (30:70 v/v (%)). Chromatographic separations were achieved reversed phase column (Merck, Purospher RP-C18, 30 ´ 4.6 (mm), 3 μm) using a mobile phase consisting of 0.1% formic acid solution and acetonitrile (85:15 v/v (%)), flow rate 0.50 (mL/min). Detection utilized a tandem MS/MS, the analytes were ionized using an ESI source in the positive ion mode prior to detection by Multiple Reaction Monitoring (MRM) mode. The analytes were monitored at the following transitions (m/z) 396.10 → 226.90, and (m/z) 348.24 → 158.10 for cefdinir and cephalexin respectively. The proposed method was fully validated in terms of linearity, accuracy, precision, specificity, sensitivity, recovery and stability, giving results within the acceptable range.


Cefdinir, HPLC-MS/MS, Plasma, ESI Source, Positive Ion Mode, MRM Mode

1. Introduction

Cefdinir (C14H13N5O5S2, f.wt. 395.42 g/mol) shown in the structure below, is (6R-[6α,7β(Z)]-7[[2-amino-4- thiazolyl)(hydroxyimino)acetyl]amino]-3-ethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid). Cef- dinir was first synthesized in 1988 and approved by the USFDA in 1997 as a third generation cephalosporin with an expanded spectrum of antibacterial activity for oral administration [1] . This encompassed pathogens that cause respiratory tract or skin and skin structure infections. In addition to being well tolerated, cefdinir demonstrated high efficacy and superior taste compared to other cephalosporins [2] . It was therefore, suggested for the treatment of pediatric patients in addition to adults and adolescents having specific mild to moderate respiratory tract or skin infections [2] . Previous studies indicated that cefdinir is excreted primarily viakidneys [3] [4] . Ma- ximal plasma cefdinir concentrations occur 2 - 4 hours post dose following capsule or suspension administration, with an estimated absolute bioavailability of about 25% [2] was demonstrated.

Structure of Cefdinir

Several high performance liquid chromatographic method, coupled with UV detection have been described for the analysis of cefdinir in human plasma, among others, these have include determination of cefdinir and related substances [5] [6] , isolation and structural elucidation of cefdinir impurities [7] [8] , distribution of cefdinir in serum and pulmonary compartment [9] , pharmacokinetic interaction between cefdinir and two angiotensin- converting enzyme inhibitors in rats [10] or the determination of cefdinir in different dosage forms using HPLC coupled with UV [11] .

In spite of the apparent benefits of cefdinir compared other cephalosporins, few reports appeared in the literature describing bioequivalence, pharmacokinetics or comparative bioavailability studies. This may be attributed to 1) lack of selectivity and sensitivity 2) analytical complications pertaining to elution of latent plasma peaks measured at the suggested wavelength when UV detection was employed, and 3) long analysis time. Until to date, only one HPLC-MS/MS method have been published for the determination of cefdinir in human plasma [1] .

The present work reports a selective, simple and sensitive HPLC-MS/MS bioanalytical method for the determination of cefdinir in human plasma.

2. Experimental

2.1. Chemicals and Reagents

Cefdinir (98.7% purity) and cephalexin (100.23% purity) were provided by pharma international (Amman-Jordan). HPLC acetonitrile gradient grade solvent, deionized water and formic acid were purchased from Merck (Darm- stadt-Germany). Reagents were used without further purification. Blank human plasma samples have been obtained from the plasma of participants who volunteered in blood bank.

2.2. Instrumentation

The LC-MS/MS system consisted of a High Performance Liquid Chromatography (Agilent 1200 Series, Agilent Technologies, Germany) coupled with a Sciex Triple Quadrupole Mass Spectrometer (API 5000, MDS, Sciex, Ontario-Canada) equipped with an electrospray ionization (ESI) (Applied Biosystems). Data acquisition and processing were controlled by Applied Biosystems/MSD SCIEX Analyst software (Version 1.4.2).

2.3. Chromatographic Conditions and MS/MS Instrumental Settings

Chromatographic separations were performed using Merck, Purospher RP-18e column (30 mm ´ 4.6 mm, 3 μm) thermostated at 40˚C. The mobile phase was an aqueous solution of formic acidin water (0.1%) and acetonitrile (85:15, v/v (%)). The separation was performed under isocratic conditions set at a constant flow rate of 0.50 (mL/min). The injection volume was fixed at 20 µL.

LC-MS/MS experimental conditions utilized the Multiple Reaction Monitoring (MRM) for both cefdinir and the internal standard (cephalexin). The detection was performed in the positive ESI mode for each of the analytes respective ions [M + H]+. Instrument settings of the MS/MS are summarized in Table 1. The fragmentation pattern of cefdinir and cephalexin are shown (A and B) (Figure 1), whereas the full scan product ion spectrum of [M + H]+ of cefdinir is illustrated in Figure 2.

2.4. Preparation of Stock and Working Standard Solutions

A cefdinir stock solution was prepared by dissolving of 25.00 mg cefdinir in water (100.0 mL) to make up the concentration of 250.0 (µg/mL). Two working standard solutions concentration of 25.00 and 1.00 (μg/mL) were also prepared. The internal standard (cephalexin) stock solution was prepared by dissolving of 20.00 mg cephalexin in a 100.0 mL of formic acid in water (0.1%) with acetonitrile (70:30 v/v (%)) to make up the concentration 200.0 (µg/mL).

Table 1. Experimental setting for the tandem mass-spectrometer during the analysis of cefdinir and cephalexin (IS).

(a) (b)

Figure 1. The fragmentation of (a) cefdinir and (b) cephalexin.

Figure 2. Full scan product ion spectrum of [M + H]+ of cefdinir.

2.5. Preparation of Matrix Based Calibrators and Quality Control Samples

Cefdinir matrix based calibrators were prepared from the above working standard solutions of cefdinir in 5.0 mL plasmato produce the following calibrators: 10.0, 50.0, 75.0, 150.0, 325.0, 500.0, 675.0, 850.0, and 1200.0 (ng cefdinir/mL). Additionally 10.0 mL standard solutions of each of the quality control (QC) samples were prepared in blank plasma to make up the concentrations of: 30.0, 600.0, and 960.0 (ng/mL). Prior to analysis each sample (250 µL) was spiked with a 200 ng aliquot of cephalexin.

2.6. Sample Preparation

To each plasma aliquot (250 µL) in an eppendorf tube (1.5 mL capacity),a 1.0 mL of a mixture of 0.1% formic acid in water with acetonitrile (30:70 (v/v) %) solution containing the IS (200 ng/mL) was added, samples were then vortexed for (30 s) and centrifuged (4000 rpm per min) for five min. A 200 µL aliquot of the supernatant was transferred to a 10 ml glass tube. The mixture was then diluted to a total volume of 4.00 mL. The solution was vortexed for (30 s) before its transfer to the well plate of the autosampler (200 µL). A 20 µL volume was injected onto the equilibrated LC-MS/MS system.

3. Results and Discussions

3.1. Method Validation

The developed method was fully validated with respect to the following parameters: selectivity, stability, linear- ity, limit of detection and as well as of lower and upper limits of quantitation, sensitivity, recovery, accuracy, and precision.

3.1.1. Selectivity

All samples were extracted and analyzed using the developed and optimized method, no interferences were observed at the retention times of cefdinir or the IS. Analytical signals from blank plasma sample extracts and from spiked samples at the lower limit of quantitation (10 ng/mL) are illustrated in Figure 3. In addition, chromatograms obtained from incurred samples after 3 h dosing with suspension (125 mg/mL Cefdinir) are illustrated in Figure 3. A high selectivity with no matrix effects were observed, this is attributed to the high dilution factor (20) of the protein precipitated matrix. The use of a highly sensitive MS/MS (API 5000) facilitated the recording

(a) (b)

Figure 3. Representative chromatograms of (a) cefdinir and IS (cephalexin, b) in human plasma samples. (I) Blank plasma sample; (II) Blank plasma sample spiked with cefdinir at BLLOQ 10 ng/mL and IS; and (III) Plasma sample from a volunteer 3 hour after administration of 125 mg/5mL suspension of cefdinir.

of a high signal. Consequently a simple, highly selective and highly sensitive method was concluded.

3.1.2. Linearity, Linear Working Range and Calibration Model

Response function of cefdinir was plotted against corresponding concentration level in the dynamic ranges: 10 to 1200 ng/mL. Linearity for six calibration curves, was demonstrated by visual inspection and by calculating the correlation coefficient, of 0.9976 using the weighted regression model with a statistical weight of, the calibration equations were y = 0.001026C + 0.000218, where y represents peak area ratio of analyte to IS and X represents the analyte concentration. The lower limit of quantitation for cefdinir was found to be 10 ng/mL.

3.1.3. Precision and Accuracy

Six replicates measurements of each quality control containing QCL 30 (ng/mL), QCM 600 (ng/mL) and QCH 960 (ng/mL) matrix based standards of cefdinir were chromatographed to evaluate instrument precision, as well as method, inter-day, and intra-day precision and accuracy. The results summarized in Table 2. Results indicate that the bioanalytical method id precise not exceeding 20% at LLOQ and not exceeding 15% for all other matrix based calibrators.

3.1.4. Stability

Stability during sampling, sample storage, processing and analysis was investigated. Stability data was evaluated

with respect to analytical signals obtained from freshly prepared QC samples, compared to those samples meas- ured after stressed conditions. Stability experiments extended throughout the analysis duration until assay of the last harvested sample. For short term stability studies, quality control samples in plasma including QCL (30 ng/mL), QCM (600 ng/mL) and QCH (960 ng/mL) were thawed and kept un-treated, at room temperature for 6 h. Autosampler stability was evaluated over 65 h. Freeze and thaw stabilities covered five cycles of freeze and thaw cycles. Long-term matrix based solution stability was investigated under prolonged storage condition (198 days, −80˚C). Table 3 which summarizes stability data demonstrate that cefdinir was stable under the investigated experimental conditions.

3.2. Method Application

The validated bioanalytical method is recommended to apply and evaluate the comparative bioavailability (bioequivalence) of a test and reference drug products for cefdinir (125 mg/5mL, a paediatric suspension) in healthy volunteers in the fasted state.

4. Conclusion

A highly sensitive, simple and fast RP-LC-ESI-MS/MS method for the determination of cefdinir in human plasma was developed and fully validated according to the current FDA guidance. This method involves a single step liquid-liquid extraction, using cephalexin, a commercially available substance, as internal standard. The

Table 2. Summary of instrument, inter-day, intra-day precision and accuracy data.

QCL: Quality control sample of low concentration; QCM: Quality control sample of medium concentration; QCH: Quality control sample of high concentration.

Table 3. Summary of stability data pertaining to cefdinir.

*Average values of peak area.

short run time of 3.0-min and the relatively low flow rate (0.50 mL/min) allows the analysis of a large number of samples with less mobile phase consumption. Validation results show that the optimized RP-LC-ESI-MS/MS method possesses specificity, accuracy, precision, sensitivity, linearity, recovery, and stability over the entire range of significant therapeutic plasma concentrations. The proposed method is recommended for the analysis of a large number of authentic plasma samples withdrawn from normal volunteers participating in a bioequivalence study.


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