A validated reversed phase HPLC assay for the determination of metronidazole in human plasma

www.wjpps.com Vol 3, Issue 12, 2014.

Hammami et al. World Journal of Pharmacy and Pharmaceutical Sciences

A VALIDATED REVERSED PHASE HPLC ASSAY FOR THE

DETERMINATION OF METRONIDAZOLE IN HUMAN PLASMA

Nada H. Binhashim, Syed N. Alvi, and Muhammad M. Hammami*

Clinical Studies and Empirical Ethics Department, King Faisal Specialist Hospital &

Research Center, MBC-03, P.O. Box 3354, Riyadh 11211, Kingdom of Saudi Arabia

ABSTRACT

A simple and precise reversed-phase high performance liquid

chromatography (HPLC) method for the determination of

metronidazole in human plasma was developed and validated. Using

tinidazole as an internal standard (IS), separation was achieved on

Symmetry shield RP18 column. The mobile phase, sodium acetate 0.05

M (pH=4, adjusted with phosphoric acid), and acetonitrile (85:15,v:v)

delivered at flow rate 1.0 ml/min. 0.25ml plasma samples were

deproteinized with methanol containing 2% perchloric acid and

centrifuged. 100µl supernatant clear solution was injected to HPLC

system. The eluent was monitored spectrophotometricly at 320 nm. No

interference in blank plasma or of commonly used drugs was observed.

The relationship between the concentration of metronidazole in plasma

and peak area ratio of metronidazole to the IS was linear over the range

of 0.05-15.0 μg/ml. Intra-day and inter-day coefficient of variation

(CV) and bias were ≤ 6.2% and ≤ 9.0%, and ≤7.3% and ≤ 11.0%,

respectively. Mean extraction recovery of metronidazole and the IS

from plasma samples was ≥ 88% using the method, metronidazole was

found stable under various conditions generally encountered in the clinical laboratory (≥93%

and ≥90% in processed and unprocessed samples, respectively). Further, the method was

successfully employed to measure metronidazole levels in plasma samples from healthy

volunteers.

KEY WORDS: Metronidazole, Tinidazole, Human plasma, HPLC.

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*Correspondence for

Author

Muhammad M.

Hammami*

Clinical Studies and

Empirical Ethics

Department, King Faisal

Specialist Hospital &

Research Center, MBC-03,

P.O. Box 3354, Riyadh

11211, Kingdom of Saudi

Arabia

[email protected],

[email protected],

Article Received on

22 September 2014,

Revised on 13 October 2014,

Accepted on 04 November 2014

http://www.wjpps.com/

mailto:[email protected]

mailto:[email protected]



INTRODUCTION

Metronidazole (CAS: 443-48-1)[2-methyl-5-nitro-1H-imidazol-1-ethanol]is an oral synthetic

antiprotozoal and antibacterial agent.It is widely used in the treatment of infections that are

proven or strongly suspected to be caused by anaerobic bacteria. [1-2]

Its oral bioavailability is

about 20%, with a mean peak plasma concentration of 4.1- 6.4µg/ml at about 1-2 hours after

one 250 mg oral dose. [3-4]

Several analytical methods have been reported for the determination of metronidazole in

pharmaceutical formulations.[5-7]

However, metronidazole level in human plasma has been

mainly determined by high performance liquid chromatography (HPLC),[8-10]

with some of the

reportedmethods using relatively large plasma volume or multiple-step extraction procedures.

[11-12]Recently, liquid-liquid and solid phase extraction (SPC)high performance liquid

chromatography-mass spectrometry(HPLC-MS/MS) techniques[13-14]

were applied in

bioequivalence and pharmacokinetic studies of metronidazole. Although,HPLC-MS/MS

methods aresimple and convenient, they areexpensive and may not be feasible for low budget

laboratories.

In the present study, we describe a simple, precise, rapid, and low-cost HPLC assay that

requires 0.25 ml human plasma, and is based on protein precipitation. The method was fully

validated and successfully applied to determine metronidazole level in plasma samples from a

healthy volunteer and to determine thestability of metronidazole under various laboratory

conditions.

MATERIAL AND METHODS

Apparatus

Chromatography was performed on a Waters Alliance HPLC 2695 (Waters Associates Inc.,

Milford, MA, USA) consisting of a quaternary pump, autosampler, column thermostat, and

photodiode array detector. A reversed-phase column Symmetry Shield RP-18 (4.5 x 150 mm,

5-μm) and a guard pak pre-column module with a Nova-pak C18 4-μm insert were used for

the separation. Data were collected with a Pentium IV computer using Empower

Chromatography Software.




Chemical and reagents

All reagents were of analytical-reagent grade unless stated otherwise. Metronidazole and

tinidazole were purchased from Sigma-Aldrich Co., St. Louis, MO, USA. Acetonitrile and

methanol (both HPLC grade), sodium acetate, and perchloricacid were purchased from Fisher

Scientific, Fairlawn, NJ, USA. HPLC grade water was prepared by reverse osmosis and was

further purified by passing through a Synergy Water Purification System (Millipore, Bedford,

MA, USA).Drug-free human plasma was obtained from the blood bank of King Faisal

Specialist Hospital & Research Centre (KFSHRC) Riyadh, Saudi Arabia after approval of the

Research Ethics Committee.

Chromatographic conditions

The mobile phase was composed of 0.05 M sodium acetate (pH adjusted to 4.0with

phosphoric acid), and acetonitrile (85:15, v:v). Before delivering into the system, the mobile

phase was filtered through 0.45 μm polyetersulfone membrane and sonicated under vacuum

for 5 minutes. The analysis was carried out under isocratic conditions using a flow rate of 1.0

ml/min at ambienttemperature and a run time of 8minutes. A photodiode array detector set at

320nm was used.

Preparation of standard and quality control samples

Stock solutions of metronidazole and tinidazole (1.0 mg/ml) were prepared in water. They

were diluted with blank human plasma and mobile phase,respectively,to produce working

solutions of 20μg/ml and100 μg/ml, respectively. Nine calibration standards in the range of

0.05 – 15.0μg/ml and four quality control (QC) samples (0.05, 0.15, 7.5, and 13.5μg/ml) were

prepared in human plasma. Calibration standards and QC samples were vortexed for one

minute and 0.25 ml aliquots were transferred into micro centrifuge tubes and stored at -20 ºC

until used.

Sample preparation

Aliquots of 0.25 ml of calibration standards or QC samples were allowed to equilibrate to

room temperature. To each tube, 40 μl of the IS working solution was added and the mixture

was vortexed for 10 seconds. 25 µlmethanol containing 2% perchloric acid were added and

samples were vortexedagain for thirty seconds. After centrifuged for 15 min at 13600 rpm,

samples were allowed to coolat -30 ºCfor 10 minutes. After a second centrifugation for 15

minutes, 100 μl supernatant clear solution was collected and injected into the HPLC system.




Stability studies

A total of 40 aliquots of QC samples(0.15, and 13.5 μg/ml)were used for stability studies.

Five aliquots of each QC sample were extracted and immediately analyzed (baseline), five

aliquots were allowed to stand on the bench-top for 24 hours at room temperature before

being processed and analyzed (counter stability, 24 hours at room temperature), five aliquots

were stored at –20 °C for fifteen weeks before being processed and analyzed (long term

freezer storage stability), and five aliquots were processed, reconstituted, and stored at room

temperature for 24 hours or 48 hours at –20 °C before analysis (autosampler stability).

Finally, fifteen aliquots of each QC sample were stored at –20 °C for 24 hours. They were

then left to completely thaw unassisted at room temperature. Five aliquots of each sample

were extracted and analyzed and the rest returned to –20 °C for another 24 hours. The cycle

was repeated three times (freeze-thaw stability).

Method validation

The method was validated according to standard procedures described in the US Food and

Drug Administration (FDA) bioanalytical method validation guidance.[15]

The validation

parameter included: specificity, linearity, accuracy, precision, recovery and stability.

RESULTS AND DISCUSSION

Optimization of chromatographic conditions

Under the optimal experimental conditions, consisting of mobile phase of sodium acetate

0.05 M (pH=4, adjusted with phosphoric acid) and acetonitrile (85:15 v/v) delivered at flow

rate 1.0 ml/min, metronidazole, tinidazole, and plasma components exhibited a well-defined

chromatographic separation within 8 minutes run. The retention times of metronidazole and

tinidazole (IS) were around 3.7 and 6.5 respectively.

Specificity

Specificity is the ability of an analytical method to differentiate and quantify the analyte in

the presence of other components in the sample. No endogenous component co-eluted with

metronidazole or the IS. Figure 1 depicts a representative chromatogram of drug free human

plasma used in preparation of standards and QC samples.

Linearity, Accuracy and Precision

Linearity of metronidazole was evaluated by analyzing ten curves of ten standard

concentrations over the range (0.05-15.0μg/ml) prepared in human plasma. Figure 2




represents an overlay of chromatograms of extracts of 0.25 ml human plasma spiked with the

IS and one of ten concentrations of metronidazole. The peak area ratios were subjected to

regression analysis(regression equation, Y = 0.102 X + 0.006). The suitability of the

calibration curves was confirmed by back-calculating the concentration of metronidazole in

human plasma from the calibration curves (Table 1). All calculated concentrations were well

within the acceptable limits. Precisionand bias were also determined for four QC

concentrations (0.05, 0.15, 7.5, and 13.5μg/ml). The intra-day (n=10) and inter-day (n=20,

over 3 days) precision was 6.2% and 7.3%, respectively.The intra-day and inter-day bias

was in the range of 2.8-9.0% and 3.1-11%, respectively. The results are summarized in Table

2.

Recovery

The absolute recovery of metronidazole was assessed by direct comparison of peak areas

from plasma and mobile phase samples, using five replicates for each of four QC

samples(0.05, 0.15, 7.5, and 13.5μg/ml). Similarly, the recovery of the IS was determined by

comparing the peak areas of the IS in 5 aliquots of 0.25 ml human plasma spiked with 40μl of

IS (100μg/ml) with the peak areas of equivalent samples prepared in mobile phase. The

results are presented in Table 3. The mean recovery of metronidazole and the IS were

88%and 94%, respectively.

Robustness

The robustness of a method is a measure of its capacity to remain unaffected by small

variations in chromatographic conditions. It gives an indication of the reliability of the

method during routine applications. The robustness of the current assay was evaluated by

altering the strength of sodium acetatebuffer (±0.01 M), pH(±0.02%), and the proportion of

methanol (±2%) in the mobile phase. No significant changes in peak area, peak shape, or

retention time were observed; indicating the robustness of the method.

Stability

Stability of analyte in biological matrices is an important pre-analytical variable. It is

necessary to perform stability studies of the analyte and IS to determine the range of

appropriate conditions and time of storage. Metronidazole and IS stability in processed and

unprocessed plasma samples(0.15, and 13.5μg/ml) was investigated. Metronidazolewas stable

in processed samples for at least 24 hours at room temperature (≥ 96%) or 48 hours at -20




°C(≥ 93%).Metronidazole in unprocessed plasma samples was stable for at least fifteen

weeks at -20 °C(≥ 90%), 24 hours of room temperature (≥ 99%), and after three freeze-and

thaw cycles (100%).

Application to a volunteer sample

Figure 3 depicts an overlay chromatogram of samples collected from a volunteer before and

after 2.0 hours ingestion of a single oral dose of 250 mg metronidazole. The measured

concentrations of metronidazole were zero and 5.36μg/ml, respectively.

Table 1: Back-calculated metronidazole concentrations from ten calibration curves

Nominal

Level

(µg/ml)

Calculated Level

(µg/ml)

CV (%) Bias (%)

Mean SD

0.05 0.056 0.003 4.7 12.5

0.1 0.106 0.006 5.7 6.1

0.3 0.291 0.028 9.7 -2.9

0.6 0.602 0.037 6.2 0.3

1.0 0.940 0.047 5.0 -6.0

3.0 2.883 0.137 4.7 -3.9

6.0 5.823 0.155 2.7 2.9

12 11.649 0.315 2.7 -2.9

15 15.253 0.255 1.7 1.7

SD, standard deviation. CV, standard deviation divided by mean measured concentration x100.

Bias, measured level - nominal level divided by nominal level x 100.

Table 2: Intra - and inter-day precision and bias of metronidazole assay

Nominal

Level

(µg/ml)

Measured Level

(µg/ml) CV (%) Bias (%)

Mean SD

Intra-day (n=10)

0.05 0.054 0.003 6.2 9.0

0.15 0.156 0.005 3.1 3.8

7.5 7.712 0.369 4.8 2.8

13.5 13.927 0.641 4.6 3.2

Inter-day (n=20)

0.05 0.056 0.004 7.3 11.0

0.15 0.155 0.007 4.2 3.1

7.5 7.747 0.365 4.7 3.3

13.5 14.023 0.572 4.1 3.9

SD, standard deviation. CV, standard deviation divided by mean measured concentration x100.

Bias, measured level - nominal level divided by nominal level x 100.




Table 3: Recovery of metronidazole and the internal standard from 0.25 ml

of human plasma

Concentration

(μg/ml) Human Plasma* Mobile Phase*

Recovery

(%)

Metronidazole

0.05

237256 288272 82

0.15

251563 298323 84

7.5 304744 326652 93

13.5 299103 327054 91

Internal standard

10 2387834 2527335.8 94

* Mean peak area (SD), n = 5.

Table 4: Stability of metronidazole under various clinical laboratory conditions

Nominal Unprocessed Processed Freeze-Thaw

Level 24 hrs 15 wks 24 hrs 48 hrs Cycle

(μg/ml) RT -20 C RT -20 C 1 2 3

0.15 112 96 96 93 109 107 102

13.5 99 90 98 100 108 103 102 Stability (%) = mean measured concentration (n=5) at the indicated time divided by mean

measured concentration (n=5) at baseline x 100. Spiked plasma samples were processed and

analyzed immediately (baseline, data not shown), after 24 hours at room temperature (24 hrs,

RT), after freezing at –20ºC for 15 weeks (15 wks, –20 ºC), or processed and then analyzed

after storing for 24 hours at room temperature (24 hrs, RT) or 48 hours at –20 ºC (48 hrs, -20

ºC); or after 1 to 3 cycles of freezing at –20ºC and thawing at RT (Freeze -Thaw).

Fig. 1Representative chromatogram of metronidazole and tinidazole-free human

plasma. The arrows indicate the retention times of metronidazole (3.7 min) and

tinidazole (internal standard, IS)(6.5 min).




Fig. 2Overlay of chromatograms of extracts of 0.25 ml human plasma spiked with the

internal standard (IS) and one of tenconcentrations of methotrexate, 0.0, 0.05, 0.1, 0.3,

0.6, 1.0, 3.0, 6.0, 12.0, and 15.0 μg/ml.

Fig. 3An overlay of chromatograms of plasma samples obtained from a healthy

volunteer before (A) a nd 2.0 h after (B) a single oral 250 mg metronidazole dose.

CONCLUSION

The described HPLC assay is accurate, precise, and rapid. It requires only 0.25 ml plasma

and utilizes a simple and convenient method for sample preparation. The assay was applied to

monitor stability of metronidazole under various conditions generally encountered in the




clinical laboratories. Further, it was successfully applied to determine levels of metronidazole

in samples obtained from a healthy volunteer.

ACKNOWLEDGEMENT

This work was funded by a grant to Dr. Muhammad M Hammami, from the King Abdul-Aziz

City for Science and Technology, Riyadh, Saudi Arabia (National Comprehensive Plan for

Science and Technology # 10-BIO961-20).

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A validated reversed phase HPLC assay for the determination of metronidazole in human plasma