ISSN 1061-9348, Journal of Analytical Chemistry, 2008, Vol. 63, No. 9, pp. 881–887. © Pleiades Publishing, Ltd., 2008.
ARTICLES
Simultaneous Quantification of Cefpirome and Cetirizine or Levocetirizine in Pharmaceutical Formulations and Human Plasma by RP-HPLC 1 M. S. Araynea, N. Sultanab, and M. Nawaza a
b
Department of Chemistry, University of Karachi, Karachi–75270, Pakistan Research Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Pakistan e-mail:
[email protected] Received March 6, 2007; in final form, November 21, 2007
Abstract—A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the simultaneous determination and quantification of cefpirome and cetirizine or cefpirome and levocetirizine in pharmaceutical formulations and human plasma without changing the chromatographic conditions is described. Chromatographic separations were performed on a prepacked Nucleosil 120, C18 (5 µm, 12.5 ± 0.46 mm) column using CH3CN : H2O (75 : 25, v/v) as a mobile phase at a flow rate of 1 mL/min while UV detection was performed at 232 nm for monitoring the effluent. A number of other brands of C18 columns were also employed which had a significant effect on the separation. The method has been validated over the concentration range of 0.5–50 µg/mL (r2 > 0.999). The limit of detection (LOD) and quantification (LOQ) for cefpirome and levocetirzine in pharmaceutical formulations and serum were in the range 0.24–1.31 µg/mL. Analytical recovery from human plasma was >98%, and the within and between-day relative standard deviation was
0.999) for each of the analytes over their calibration ranges. The linearity was evaluated by the least-squares regression method and the proposed method was evaluated by its correlation coefficient and intercept value.
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SIMULTANEOUS QUANTIFICATION OF CEFPIROME
Characteristic parameters for regression equation (y = mx + c) of the HPLC method obtained by the leastsquares treatment of the results were used to confirm the linearity of the method developed. The limit of detection (LOD) for cefpirome was 0.49 and 0.37 µg/mL in the pharmaceutical formulation and serum, respectively; for lecocetirizine, it was 0.24 and 0.65, respectively. The quantification limit (LOQ) for cefpirome in the pharmaceutical formulation and serum was 0.37 and 1.09 µg/mL, respectively; for levocetirizine, it was 0.45 and 0.91 µg/ml at concentrations where the signal-to-noise ratios were equal to 3 and 10, respectively. The calculated LOD and LOQ values confirmed that the methods were sufficiently sensitive.
Table 3. Within-day and between-day accuracy and precision for the determination of cefpirome and levocetirizine in pharmaceutical formulation Nominal Mean found concentration, concentration, µg/mL µg/mL
0.5
0.49
99.61
3.45
1.00
0.98
98.10
1.75
2.00
1.97
98.92
1.55
4.00
3.98
99.53
1.42
5.00
5.01
100.30
2.34
10.00
9.80
98.03
1.08
50.00
50.09
100.38
1.36
0.5
0.49
99.59
3.26
1.00
0.99
99.85
1.46
2.00
1.96
98.89
1.27
4.00
3.99
99.87
1.38
5.00
4.97
98.99
1.80
10.00
10.01
100.21
2.05
50.00
49.96
99.94
1.10
Levocetirizine Within-day
The method ruggedness was demonstrated using two analytes by an assay on separate lots of cefpirome and levocetirizine. Each analyst prepared samples in triplicate and used separate instruments, reagents, diluent, and mobile phase solutions. The RSDs (n = 4) for all of the samples for each lot were less than 3.0%, indicating an acceptable ruggedness.
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Precisionb, RSD, %
Between-day
No change was observed in the chromatogram of levocetirizine in the presence of common excipients. The chromatogram of a pure drug sample was matched to the marketed formulation. The specificity was also determined by screening five different batches of a controlled human plasma and also along with the formulation. There was no interference of the plasma peaks in the drug–plasma samples. Figures 4b and 4c represent chromatograms of the blank plasma and the plasma containing both drugs, respectively. Therefore, the proposed method is specific and selective for the drug.
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Accuracya, %
Cefpirome Within-day
Within-day precision (repeatability) was determined by calculating the relative standard deviation (RSD %) for four determinations at each concentration of the samples and it was found to be less than 4%. Between-day precision and accuracy were assessed by assaying samples in triplicate on three separate occasions. The assay precision was 4.0% based on RSD values (RSD %). Assay accuracy, assessed by calculating the estimated concentrations as a percentage of the nominal concentrations, was better. Moreover, the RSD (less variation) showed a good precision for the developed HPLC method (Tables 3–5).
A specific, sensitive, rapid, and precise HPLC assay for the simultaneous determination of cefpirome and cetirizine or levocetirizine was developed and validated. The method is suitable for the analysis of drugs in pure form, dosage formulations, and plasma samples. The retention time observed (less than 5 min) allows for the rapid determination of the analytes, which is important for quick analysis. The method provides a satisfactory accuracy and precision with low limits of detection and quantification. Thus, the method was successfully applied to assaying the commercial cefpirome, cetirizine, and levocetirizine in individual formulations.
885
0.5
0.49
99.61
2.76
1.00
0.98
98.10
2.05
2.00
1.97
98.92
1.85
4.00
4.02
100.10
1.75
5.00
4.98
99.79
1.35
10.00
9.80
98.03
1.91
50.00
49.98
99.88
1.14
Between-day 5.0
0.48
98.86
2.85
1.00
0.99
99.95
2.31
2.00
1.98
99.94
1.95
4.00
3.99
99.75
1.63
5.000
4.97
99.92
2.21
10.00
9.87
98.45
1.56
50.00
50.05
100.02
1.82
Note:
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a
Accuracy: found concentration expressed in % of the nom inal concentration. b RSD: relative standard deviation.
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ARAYNE et al.
Table 4. Within-day percent accuracy (recovery) of the method for plasma drug analysis Conc. Injected (µg/mL)
Found (meana)
CV, %
Accuracy, %
Found (meana)
CV, %
Accuracy, %
0.50 1.00 2.00 4.00 5.00 10.00 50.00
0.489 0.999 1.997 4.017 5.008 10.033 49.085
0.016 0.001 0.001 0.003 0.001 0.002 0.013
97.80 99.90 99.85 100.43 100.16 100.33 98.17
0.491 0.984 2.012 3.982 5.017 10.005 50.069
0.013 0.011 0.004 0.003 0.002 0.000 0.001
98.20 98.40 100.60 99.55 100.34 100.05 100.14
Note: a Mean found values represent “concentration mean” of six different samples for each concentration.
Table 5. Between-day precision and accuracy for the determination of cefpirome and levocetirizine in human plasma Conc. added, µg/mL Cefpirome 0.50 1.00 2.00 4.00 5.00 10.00 50.00 Levocetirizine 0.50 1.00 2.00 4.00 5.00 10.00 50.00
Recovered concentration Day 1
Day 2
Day 3
Day 4
mean recovered
0.50 1.00 2.00 4.00 5.01 9.96 50.04
0.49 1.00 2.00 4.00 5.03 10.00 49.83
0.50 1.00 2.01 3.90 5.03 10.16 49.90
0.49 1.02 2.01 4.00 5.00 10.11 49.70
0.49 1.00 2.01 3.97 5.02 10.06 49.92
1.291 1.156 0.289 1.269 0.335 0.903 0.316
98.80 100.40 100.28 99.29 100.36 100.59 99.83
0.50 1.00 2.00 4.00 5.01 10.00 49.94
0.49 1.00 1.99 3.98 4.96 9.98 49.99
0.48 0.98 1.98 3.95 4.88 9.97 49.84
0.48 0.97 1.97 3.94 4.85 9.95 49.89
0.49 0.99 1.99 3.97 4.92 9.97 24.95
1.954 1.259 0.593 0.667 1.453 0.197 0.148
97.25 98.83 99.36 99.18 98.50 99.73 99.90
CV, %
accuracy, %
Note: CV % is coefficient of variation.
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