Rapid Determination of Metronidazole and 2-Hydroxymetronidazole in Murine Blood Plasma

Abstract

Metronidazole is a drug used to treat bacterial and protozoan infections. Nowadays, it is one of the most frequently prescribed drugs worldwide. The main aim of this paper is to present a rapid, reliable and simple high-performance liquid chromatography (HPLC) method to determine metronidazole along with its primary metabolite, 2-hydroxymetronidazole, in plasma or serum using paracetamol as an internal standard. A total of 100% methanol was used to denature plasma proteins. After centrifugation, the supernatant was evaporated under nitrogen flow. The samples were dissolved in the mobile phase and injected into a Li-Chrospher RP-18 column. A total of 10 mmol/L NaH₂PO₄: acetonitrile (90:10, v/v) solution with a flow rate of 1 mL/min was used as the mobile phase. Metronidazole and 2-hydroxymetronidazole were detected at two different wavelengths at 320 nm and 311 nm, respectively. The method is characterized by high precision (relative standard deviation % < 6). The method was used for the determination of metronidazole and 2-hydroxymetronidazole in murine blood using small amounts of plasma (≤100 μL).

Introduction

Metronidazole (1-[2-hydroxyethyl]-2-methyl-5-nitroimidazole), a synthetic derivative of azomycin (Figure 1), is an antibacterial and antiprotozoal drug. In the 1960s, metronidazole was initially used for the treatment of trichomoniasis in human. Since then, it has been used against various bacterial and protozoan infections, such as Helicobacter pylori, Entamoeba histolytica and Giardia lamblia (1). It is also used in the treatment of oral and dental infections, bone and joint infections, endocarditis, septicemia and respiratory tract infections (2). Metronidazole is a prodrug and needs to be activated in bacteria. A molecule of metronidazole is converted by reduction to a nitroso free radical, a cytotoxic form of the drug, which can subsequently interact with bacterial DNA nonspecifically, leading to DNA breakage and degradation (3). Metronidazole is well absorbed when given orally; its plasma concentration peak is reached between the first and second hour after administration. Active metabolites can be found in plasma (4). Metronidazole, like the majority of xenobiotics, is metabolized in the liver, undergoing a hydroxylation to 2-hydroxymetronidazole (Figure 1) (which is also the primary metabolite found in urine), as well as an oxidation to 1-metronidazole acetic acid and glucuronide conjugation (5). From the previous literature review, no rapid and simple HPLC assay has been developed for the determination and quantification of metronidazole and 2-hydroxymetronidazole in small amounts of plasma (40–100 μL). Nowadays, the LC/MS techniques predominate (6), as high-throughput UPLC–MS/MS methods are sensitive and they can use even smaller amounts of plasma. For example, Stancil et al. (7) developed a method for the quantitation of metronidazole and 2-hydroxymetronidazole using only 10 μL of human plasma. On the other hand, simple HPLC methods are generally quick, much cheaper, and instruments are easier to operate, which is an advantage for a still great number of laboratories. The main aim of this study was to provide a quick, reliable, and mainly simple HPLC method for the determination and quantification of metronidazole and its active metabolite 2-hydroxymetronidazole in murine plasma (respectively serum) in small sample amounts.

Figure 1

Structure of metronidazole (A) and its primary metabolite 2-hydroxymetronidazole (B).

Experimental

Chemicals

Metronidazole and 2-hydroxymetronidazole standards, acetonitrile of HPLC grade and all other chemicals of analytical grade were purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Reference standards used in the study (metronidazole, 2-hydroxymetronidazole and paracetamol) were all of suitable purity for HPLC analysis.

HPLC—instrument and conditions

For HPLC analysis, a Shimadzu LC-20 HPLC system (Shimadzu, Kyoto, Japan) with UV/fluorescence detection was used. The measurements were performed in a LiChrospher RP-18 column (5 μm) 250 × 4 mm, equipped with a 4 × 4 mm guard column (Merck, Darmstadt, Germany). A total of 10 mmol/L NaH₂PO₄: acetonitrile (90:10, v/v) solution was used as the mobile phase. The sample injection volume was 20 μL, using a 1 mL/min flow rate. The oven temperature was 40°C. Metronidazole was detected at 6.3 min (λ = 320 nm), 2-hydroxymetronidazole at 4.1 min (λ = 311 nm) and the internal standard (IS), paracetamol, at 4.9 min (λ = 300 nm). The time of analysis was 9 min per sample. The results were analyzed using the software LabSolutions (Shimadzu, Kyoto, Japan).

Calibration standard samples

Standard solutions of metronidazole and 2-hydroxymetronidazole (10 mmol/L final concentrations) were prepared by dissolving an accurately weighted amount of each compound in 96% ethanol. The IS was prepared by dissolving paracetamol in 96% ethanol to yield a 10 mmol/L concentration. Solutions were stored at −80°C and were stable for at least 4 months. Calibration samples were prepared by dissolving a standard solution of metronidazole or 2-hydroxymetronidazole in the mobile phase, and then the murine plasma was added to yield final concentrations of calibration standards (concentrations in the range of 1–200 μmol/L for metronidazole, 0.5–50 μmol/L for 2-hydroxymetronidazole).

Six calibration standards of spiked plasma were used for each concentration level.

Animals and plasma collection

The method of the animal experiment is a version of a method used in our previous work (8). Two-month-old specific-pathogen-free (SPF) male Balb/c mice were used. The mice lived under sterile conditions in Trexler-type plastic isolators and were fed with 50-kGy irradiated sterile pellet diet Altromin 1410 (Altromin, Lage, Germany). All animals were kept in a room with a 12 h light–dark cycle at 22°C. Metronidazole was applied as one intragastric dose (5 mg/kg) to mice. In each of the four groups, four mice were euthanized (control group without metronidazole administration and groups for plasma collection at 2, 6 and 24 h after metronidazole application); blood samples were collected from the carotid artery into EDTA-treated tubes. The blood samples were centrifuged at 3,200 × g for 10 min, and plasma was separated and stored at −80°C until use. The experiments were approved by the Committee for the Protection and Use of Experimental Animals of the Institute of Microbiology, Academy of Sciences of the Czech Republic (approval ID: 18/2019). The methods were carried out in accordance with the approved guidelines.

Figure 2

Representative chromatogram of mobile phase (A) and blank plasma (B) spiked with IS and metronidazole (MTZ). IS (dash-dotted line) was detected at 300 nm and metronidazole (solid line) at 320 nm. The analysis time was 9 min.

Sample preparation for HPLC analysis

Plasma samples obtained from SPF mice and calibration standard samples were prepared as follows: 10 μL of IS was added to each 100 μL of the sample (to a final concentration of 0.7 mmol/L in the mixture). Subsequently, 330 μL of 100% methanol was added, and each sample was vortexed for 5 sec. Samples were then centrifuged at 14,100 × g for 10 min at room temperature. The supernatant was transferred to a 1.5 mL Eppendorf tube and evaporated under nitrogen flow at 40°C. A total of 110 μL of mobile phase was added into the dry tubes, followed by vortexing the samples for 10 sec and centrifuging at 14,100 × g for 10 min at room temperature.

Results

Selectivity and specificity

Method selectivity was evaluated by comparing blank murine plasma with the solutions of the analytes and IS. After the injection of metronidazole, 2-hydroxymetronidazole and paracetamol (IS) into the column, three sharp peaks were observed. Metronidazole was detected at 6.3 min, 2-hydroxymetronidazole at 4.2 min and paracetamol at 4.9 min.

In the blank plasma, there were no peaks detected at retention times corresponding to analytes and IS (Figures 2 and 3). The evaluation of selectivity showed no potential interfering substances in the blank plasma. The specificity of this method showed the ability to detect and differentiate the analytes and IS in the plasma sample, and three sharp and well-resolved peaks were observed.

Figure 3

Representative chromatogram of mobile phase (A) and blank plasma (B) spiked with IS and metabolite 2-hydroxymetronidazole (2-OHMTZ). The 2-hydroxymetronidazole (solid line) was detected at 311 nm and IS (dash-dotted line) at 300 nm. The analysis time was 9 min.

Figure 4

Concentrations of MTZ and 2-hydroxymetronidazole (2-OHMTZ) in plasma of SPF mice at various time intervals after application. Control mice—without metronidazole administration; 2, 6 and 24 h after metronidazole administration.

Linearity

For metronidazole and 2-hydroxymetronidazole, six calibration points were prepared (concentrations—1, 5, 10, 20, 50 and 200 μmol/L for metronidazole; 0.5, 1, 5, 10, 20 and 50 μmol/L for 2-hydroxymetronidazole). Both calibration curves were constructed by linear least-squares regression analysis plotting of the peak-area ratio (metronidazole, resp. 2-hydroxymetronidazole/IS paracetamol) versus the concentration ratio of the analyte and IS The calibration curves were linear over the range of chosen concentrations for metronidazole (R = 0.99995, R² = 0.99989) and 2-hydroxymetronidazole (R = 0.99977, R² = 0.99954).

Recovery, precision and accuracy

Recovery was determined by comparing peak areas of metronidazole in spiked mobile phase and in spiked murine plasma. Blank murine plasma was spiked with metronidazole or 2-hydroxymetronidazole to a final concentration of 1, 10, 50 and 200 μmol/L (metronidazole) and 0.5, 5, 20 and 50 μmol/L (2-hydroxymetronidazole). This analysis was performed for three replicates at each concentration level. The average recoveries for metronidazole at concentrations of 1, 10, 50 and 200 μmol/L were 96.8, 94.0, 96.3 and 72.6%. The average recoveries for metabolite 2-hydroxymetronidazole at concentrations of 0.5, 5, 20 and 50 μmol/L were 89.2, 82.6, 75.8 and 95.3%.

Intra- and interday precision and accuracy measurements were determined at four different concentration levels of the QC samples. The results are presented in Table I. Satisfactory results were obtained with relative standard deviation (RSD) % < 6.

Table I

Precision and Accuracy of Calibration Measurements in Murine Spiked Plasma (with Metronidazole and 2-Hydroxymetronidazole)—Intraday and Interday Assay

Metronidazole–intraday assay				2-Hydroxymetronidazole–intraday assay
Concentration (μmol/L)	Precision		Accuracy (%)	Concentration (μmol/L)	Precision		Accuracy (%)
	Mean (μmol/L) ± SD	RSD (%)			Mean (μmol/L) ± SD	RSD (%)
2.5	2.34 ± 0.043	1.81	93.77	2.5	2.17 ± 0.051	2.37	86.73
15	14.57 ± 0.191	1.31	97.11	15	14.46 ± 0.250	1.73	96.42
75	75.27 ± 0.985	1.31	100.36	75	73.20 ± 0.843	1.15	97.59
125	124.99 ± 0.881	0.71	99.99	125	119.80 ± 3.314	2.77	95.84
Metronidazole–interday assay				2-Hydroxymetronidazole–interday assay
Concentration (μmol/L)	Precision		Accuracy (%)	Concentration (μmol/L)	Precision		Accuracy (%)
	Mean (μmol/L) ± SD	RSD (%)			Mean (μmol/L) ± SD	RSD (%)
2.5	2.40 ± 0.063	2.61	95.94	2.5	2.21 ± 0.090	4.05	88.58
15	14.82 ± 0.253	1.71	98.80	15	13.55 ± 0.806	5.95	90.33
75	74.11 ± 1.900	2.56	98.81	75	70.06 ± 3.094	4.42	93.41
125	124.77 ± 1.181	0.95	99.82	125	121.38 ± 3.178	2.62	97.11

Limit of detection and limit of quantification

The limit of detection in murine plasma for metronidazole and 2-hydroxymetronidazole was 0.25 μmol/L and was determined as the analyte concentration with a signal-to-noise ratio of 3. The limit of quantification was defined as the lowest level of analyte concentration with RSD <10%, and it was 0.5 μmol/L, for both metronidazole and 2-hydroxymetronidazole.

Stability

The stability of QC samples was determined at four different concentration levels ranging from 2.5 to 125 μmol/L (Table II). Samples of murine plasma spiked with metronidazole or 2-hydroxymetronidazole were frozen at −20°C or − 80°C for 7 days. We observed no significant difference between samples with RSD ≤ 6%. Freeze–thaw stability was determined at two different levels (2.5 and 125 μmol/L) (Table III). Freeze–thaw cycles (n = 3) gave RSD ≤ 3%, except for 2.5 μmol/L metronidazole (RSD = 7.01%).

Table II

Stability of Metronidazole and 2-Hydroxymetronidazole after 7 days

	−20°C				−80°C
	Metronidazole		2-Hydroxymetronidazole		Metronidazole		2-Hydroxymetronidazole
Concentration (μmol/L)	Mean (μmol/L) ± SD	RSD (%)	Mean (μmol/L) ± SD	RSD (%)	Mean (μmol/L) ± SD	RSD (%)	Mean (μmol/L) ± SD	RSD (%)
2.5	2.50 ± 0.150	6.00	2.38 ± 0.125	5.25	2.55 ± 0.045	1.78	2.36 ± 0.048	2.05
15	15.24 ± 0.418	2.74	13.53 ± 0.205	1.52	15.23 ± 0.789	5.18	13.56 ± 0.415	3.06
75	75.03 ± 0.992	1.32	76.28 ± 1.109	1.45	74.42 ± 2.002	2.69	75.33 ± 3.318	4.40
125	126.75 ± 2.477	1.95	134.90 ± 5.813	4.31	127.06 ± 2.450	1.93	127.28 ± 1.016	0.80

Table III

Stability of Metronidazole and 2-Hydroxymetronidazole—Freeze–Thaw Cycle

	Freeze–Thaw
	Metronidazole		2-Hydroxymetronidazole
Concentration (μmol/L)	Mean (μmol/L) ± SD	RSD (%)	Mean (μmol/L) ± SD	RSD (%)
2.5	2.48 ± 0.174	7.01	2.49 ± 0.054	2.18
125	125.36 ± 1.307	1.04	131.36 ± 3.379	2.57

Matrix effect

The matrix effect was determined by analyzing three replicates of low- and high-concentration QC samples using human plasma spiked with metronidazole or 2-hydroxymetronidazole. The precision results were satisfactory, with RSD ≤ 3.5%. The accuracy was within ±15% of the nominal concentrations.

Application of the method

The validated HPLC method was successfully applied to determine metronidazole and its primary metabolite, 2-hydroxymetronidazole in murine plasma after the application of metronidazole. Murine plasma was collected after two, six and 24 h of application.

Plasma concentrations of metronidazole of SPF mice are illustrated in Figure . The plasma peak concentration of metronidazole (2 h after metronidazole administration) was 99.08 ± 35.74 μmol/L. At 6 h after application, the concentration was decreased by ~2/3 (33.09 ± 8.67 μmol/L). Interestingly, we were able to detect metronidazole even 24 h after administration (0.12 ± 0.10 μmol/L). Levels of the primary metabolite, 2-hydroxymetronidazole were noticeably lower than metronidazole. At 2 h after application, the concentration was 15.90 μmol/L ± 5.18 and at 6 h it was 10.79 μmol/L ± 1.39. After 24 h, there was no detectable concentration of 2-hydroxymetronidazole.

Discussion

There are several methods for determining metronidazole found in the literature. Kaye et al. (9) described a rapid HPLC assay using a 100 μL of biological fluid sample. Pähkla et al. (10) were able to analyze metronidazole in only 50 μL of plasma or saliva, but the method itself is time-consuming (samples after addition of acetonitrile need to be frozen overnight). Suyagh et al. (11) analyzed even smaller amounts of blood (30 μL as a dried blood spot), but the blood spots needed to be left to dry for at least 3 h. In addition, these assays only determined the metronidazole. There are also studies in the literature discussing the development of methods for the simultaneous determination of metronidazole and other medication, such as ranitidine (12), amoxicillin (13) and omeprazole (14). These combinations of drugs are used in the eradication of H. pylori. There is also a study, reported by Tan et al. (15) focusing on the pharmacokinetic interaction between metronidazole and imatinib. Although Jessa et al. (16) have developed a rapid and selective HPLC method for the determination of metronidazole and its hydroxylated metabolite, their method was not suitable for us, as there was a need for a larger sample volume—0.5 mL, and this assay could not to be implemented into small volumes of plasma, i.e., from mice. This was also the case with the method developed by Klimowicz et al. (17).

Another method published recently by Stancil et al. (7) is based on UPLC–MS/MS; an advantage of their method is small volume of sample needed (10 μL); on the other hand, the method described in our paper is reliable, quick and much easier. It allows to work with small volumes of sample as it has been used for analysis of samples of murine plasma (Figure ). In fact, using mice as experimental animals, we focused on developing a method that was able to quantify metronidazole and its metabolite in small sample volumes, which was possible by monitoring metronidazole and 2-hydroxymetronidazole simultaneously at different wavelengths (320 nm, respectively 311 nm).

Conclusions

In conclusion, we developed and described a reliable, simple and quick HPLC method for the determination of antibacterial and antiprotozoal drug metronidazole in murine plasma. The advantage of this method is the simultaneous determination of the parent compound as well as the primary metabolite of metronidazole, i.e., 2-hydroxymetronidazole. The method described is sensitive enough, and there is no need to use LC–MS-based methods (18), despite the small volumes of murine plasma samples. Extraction into an organic phase (methanol) and a small injection volume (20 μL) enable using even smaller sample volumes (40–100 μL).

Acknowledgments

This study was supported by grant 19-08294S from the Czech Science Foundation and by Internal Student Grant Agency of Palacký University IGA_LF_2020_022.