BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF NITROFURANTOIN IN HUMAN PLASMA BY LC -MS / MS

BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF NITROFURANTOIN IN HUMAN PLASMA BY LC -MS / MS

Meruva Sathish Kumar^* and P. Shanmugapandiyan

Department of Pharmacy, Mewar University, Chittorgarh - 312901, Rajasthan, India.

ABSTRACT: A simple, rapid and sensitive method using an isocratic Liquid chromatography coupled with Tandem mass spectrometry was developed and validated for the assay of nitrofurantoin in the Human Plasma. The Mass transition of nitrofurantoin and losartan (Internal standard) were M/z 237.1/151.9 and M/Z 421.300/ 179 in ESI Negative ionization. Linearity was observed between the nitrofurantoin concentration and the peak area ratio from 10.451 to 1033.897 ng/mL with R2 value of 0.99. Plasma samples containing nitrofurantoin were extracted with Acetonitrile: 5mM Ammonium Acetate (pH 3.8) (80:20). The observed recovery of nitrofurantoin was 90%. The intra-day and inter-day accuracy was performed. Stability parameter was performed. The method will be used in the determination of the pharmacokinetic parameters of nitrofurantoin.

LC-MS/MS, Nitrofurantoin, Losartan, Validation

INTRODUCTION: Nitrofurantoin is an antibiotic that has fights bacteria in the body. Chemically it is (E)-1-[(5nitro2furyl)methylideneamino] imidazolid  ine-2,4-dione ^{1 - 4}. It has an empirical formula of C₈H₆N₄O₅ and a molecular weight of 238.16 g/mol. It works as an antibiotic by damaging bacterial DNA, since its reduced form is highly reactive. It is used in the treatment of UTI’s caused by susceptible bacteria nitrofurantoin is readily absorbed after oral dosing from the gastrointestinal tract with peak plasma levels occurring in 4 - 8 hrs and  half life  is 20 - 60 minutes. Literature survey reveals that very few methods were developed and validation for the quantification of nitrofurantoin in pharmaceutical and biological fluids ^{5 - 13}.

The aim and objective of proposed method is to develop the simplest, sensitive, high recovery and selective method with proper internal standard usage. The method was validated as per ICH guidelines ¹⁴.

FIG. 1: STRUCTURE OF NITROFURANTOIN

MATERIALS AND METHODS:

Instrumentation: A thermo LC system equipped was used to inject 25μl of the samples on a Hypurity advance Kromosil-C-8; 4.6x50mm which was kept at ambient temperature of 25 °C. The Electron Spray Ionization source was heated at 4500 °C nebulizer operating in negative ion mode. A mixture of buffer and acetonitrile in the ratio of 20:80 % v/v was degassed ultrasonically for 5 min. The flow rate of mobile phase was 0.8 ml/min (split ratio-0.5ml).

Preparation of Standard Stock and Plasma Samples: The nitrofurantoin standard stock solution of 1033896.6 ng/ml was prepared by dissolving required quantity in acetonitrile and dimethyl sulphoxide. This was further diluted with acetonitrile and water to get a concentration of 51694.8 ng/ml. The spiked calibration curve standards (CC) and quality control samples (QC) were prepared by using standard stock and intermediate stock solution in 80% acetonitrile in water. The internal standard (IS) stock solution was prepared by dissolving 10 mg in methanol. This was further diluted with methanol to get a concentration of 1033896.6 ng/ml. all the solutions were stored at - 20 °C. The CC standards and QC samples were prepared by spiking human plasma with respective working solutions. CC standards were prepared at 10.4505, 26.1262, 52.2524, 104.5048, 261.2621, 466.5394, 681.0794, 878.8121 and 1033.8966 n/ml for nitrofurantoin. QC samples were prepared at 10.5163 ng/ml (LLOQ QC), 28.8119 ng/ml (LQC), 364.7070 ng/ml (MQC) and 878.8121 ng/ml (HQC) concentrations as shown in the Table 1 and 2. All the spiked samples were stored at -20 °C for sample analysis and validation.

TABLE 1: PREPARATION OF SPIKED CALIBRATION CURVE STANDARDS

TABLE 2: PREPARATION OF SPIKED QUALITY CONTROL SAMPLES

Sample Preparation: Prior to analysis, the required number of quality Control samples along with the Calibration Curve Standards was withdrawn from Ultra Low Temperature Freezer and thawed to room temperature. Thawed samples were vortexed ensuring complete mixing of contents.

500µl of samples and 50µl of Internal Standard (10000ng/ml) solution was added to all samples except blank and vortexed. add 50µl of 10% v/v Orthophosphoric acid and 2.5 ml of ethyl acetate was added to all samples and vortexed and vibramaxed at 2500 rpm for 10 minutes centrifuged at 4500rpm for 10 minutes at 4 °C in a refrigerated Centrifuge from that take 1.8 ml of supernatant samples were separated and taken in fresh ria vial were dried under nitrogen evaporator at 50 °C and 15 psi. The dried residue sample in all RIA vials was reconstituted with 0.5ml mobile phase and vortexed and injects 25μl of solution into LC-MS/MS system.

Validation Procedures: The method was validated as per US FDA guidelines. System suitability experiment was performed by injecting six consecutive injections using aqueous standard mixture of drug (LLOQ) and IS during the start of the method validation. The carryover test of the auto sampler was performed by injecting a sequence of injections consisting of standard (LLOQ, ULOQ), reconstituted solution, and standard blank and extracted standard equivalent to highest standard in the CC alternatively to check there is any carryover in the bank sample. The selectivity of the method was established by checking the blank K2 EDTA human plasma, K2 EDTA lipemic plasma and K2 EDTA haemolytic plasma obtained from 8 different donors. Also spiked six samples at LLOQ concentration of nitrofurantoin and IS in plasma of one donor from above plasmas (except hemolytic and lipemic plasmas). Compare the response of analyte and IS in blanks with the mean response of injected LLOQ. The matrix effect for the intended method was assessed by using chromatographically screened human plasma concentrations equivalent to LQC and HQC prepared with six different lots. The linearity of the method was determined by using a 1/x² weighted least square regression analysis of standard plots associated with a 9 point standard curve. The intra and inter day accuracy and precision were determined by analyzing 6 replicates of LQC, GMQC, MQC and HQC in a single day and between two consecutive days respectively. The precision (% CV) at each concentration level should not be more than 15% except for LLOQ QC where it should be 20%. The accuracy (%) must be within +15% of their nominal value except for LLOQ QC where it should be within +20%. The percentage mean recoveries were determined by measuring the concentrations of the extracted plasma QC samples at HQC, MQC and LQC against unextracted QC samples. Reinjection reproducibility was performed by analyzing 6 replicates of LQC and HQC samples. Stability results were assessed by measuring the area response of stability samples against freshly prepared LQC and HQC samples. Freeze thaw stability was estimated after 3 cycles of freezing and thawing of samples at -20 °C auto sampler stability was determined by keeping the samples in the auto sampler at 15 °C and analyzed after 36 hrs under a fresh calibration curve. Dry extract stability was evaluated by reconstituting and evaporating the samples kept on the bench at ambient temperature and analyzed after 24hrs.

RESULTS AND DISCUSSION:

Optimization of Liquid Chromatography and Mass Spectrometry Conditions: For complete chromatographic resolution of nitrofurantoin and Internal Standard, several solvents such as acetonitrile, methanol, different buffers and mixture of solvents were tried along with different flow rates. Finally the resolution of peaks was achieved with 5mM ammonium acetate (pH 3.8) in Acetonitrile: 5mM Ammonium Acetate with pH (3.8), (80:20) using High purity advance Kromosil-C-8; 4.6x50mm (25µl). The flow rate was 0.8ml/min. Following mass spectrometric conditions, precursor ion to the parent ion transitions for nitrofurantoin and IS were at m/z   237.1→151.9 and 421.3→179.0 respectively was used for quantification purpose.

Method Validation Parameters: The selectivity of the present method was established by checking any interfering compounds that elute along with nitrofurantoin. The response of analyte and IS in blanks was compared with the mean response of injected LLOQ. Hence there were no interfering peaks formed at nitrofurantoin retention time and IS retention time in the plasma blanks. Fig.1 shows representative chromatograms of K2 EDTA blank human plasma samples.

The specificity of the method was determined by comparing the response of analyte and IS with the mean response of injected LLOQ. There were no interfering peaks obtained at nitrofurantoin and IS retention time. The results were shown in Table 3.

Chromatogram of Blank Plasma and Internal Standard:

FIG. 2: SPECIFICITY AND SELECTIVITY

TABLE 3: PREPARATION OF SPECIFICITY AND SELECTIVITY

Carry Over Effect: The sequence of injections containing two blank samples and two samples containing of LLOQ and ULOQ with internal standard were analyzed alternately to check if there is any carry over affecting the blank sample. There was no carryover effect observed in the present method.

Recovery: The peak areas of extracted QC samples were compared against the peak areas of respective aqueous QC samples. The % mean recovery for cycloserine in LQC, MQC and HQC was 79.38, 74.72 and 75.12% respectively. Recovery for IS was 73.28%. The results were shown in the Table 4.

Analyte (Nitrofurantoin) Recovery:

TABLE 4A: MQC CONCENTRATION LEVEL

TABLE 4B: HQC CONCENTRATION LEVEL

TABLE 4C: LQC CONCENTRATION LEVEL

TABLE 4D: TOTAL RESULTS OF ANALYTE RECOVERY

TABLE 5A: INTERNAL STANDARD (LOSARTAN) RECOVERY

TABLE 5B: TOTAL RESULTS OF ANALYTE RECOVERY

Linearity, Accuracy, Precision and Sensitivity: The calibration curve was constructed using 9 calibration standards ranging from 10.45ng/ml to 1033.897ng/ml. a straight line fit was made through the data points by 1/x² weighing method. The correlation coefficient was found to be ≥ 0.99. The lower limit of quantification (LLOQ) was found to be 10.516ng/ml. The sensitivity results were shown in following Table 7. The percent accuracy observed for the mean of back calculated concentrations for four calibration curves for  nitrofurantoin was within 97.8-109.7, while % CV values ranged from 4.8-10.9. Accuracy and precision for intraday and inter day plasma homogenate samples are presented in the Table 6, 7 and 8. The following chromatograms represents accuracy and precision for LQC, GMQC, MQC and LQC samples. (Fig. 3, 4, 5 and 6).

FIG. 3: CHROMATOGRAM OF HQC LEVEL CONCENTRATION OF NITROFURANTOIN

FIG. 4: CHROMATOGRAM OF MQC LEVEL CONCENTRATION OF NITROFURANTOIN

FIG. 5: CHROMATOGRAM OF LQC LEVEL CONCENTRATION OF NITROFURANTOIN

FIG. 6: CHROMATOGRAM OF LLOQ QC  LEVEL CONCENTRATION OF NITROFURANTOIN

TABLE 6: SENSITIVITY OF NITROFURANTOIN

TABLE 7:  RESULTS OF PRECISION AND ACCURACY WITHIN BATCH OF NITROFURANTOIN

TABLE 8: RESULTS OF PRECISION AND ACCURACY BETWEEN BATCHES OF NITROFURANTOIN

Stability: The predicted concentrations for nitrofurantoin at LQC and HQC samples deviated with in ±15% of nominal concentration in stability tests i.e. auto sampler stability (36 hrs), bench top (6 hrs), repeated three freeze thaw cycles, 24hrs of dry extract stability. The results were found to be within the acceptance limits during the entire process (Table 9).

TABLE 9: FREEZE THAW STABILITY FOR NITROFURANTOIN QUALITY CONTROL SAMPLES QUALITY CONTROL SAMPLES

TABLE 10: BENCH- TOP STABILITY FOR NITROFURANTOIN QUALITY CONTROL SAMPLES

TABLE 11: AUTO SAMPLER STABILITY FOR NITROFURANTOIN QUALITY CONTROL SAMPLES

TABLE 12: DRY EXTRACT STABILITY FOR NITROFURANTOIN QUALITY CONTROL SAMPLES

CONCLUSION: A LC-MS/MS method was developed for the determination of nitrofurantoin in human plasma by using simple liquid - liquid extraction method. The method is simple, rapid, selective, specific, shows good accuracy and precision and cost effective  With less run time 2.5 min and ability to quantify the drug in Nanogram level This method can be used for routine analysis of determination of  nitrofurantoin in human plasma by using LC-MS/MS method

ACKNOWLEDGMENT: I thank Dr. M. Sridhar   Department of Pharmaceutical Analysis for supporting me to do this research work. I thank my guide Dr. P. Shanmugapanidyan for his immense support.

CONFLICTS OF INTEREST: Nil.

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    How to cite this article:

    Kumar MS and Shanmugapandiyan P: Bioanalytical method development and validation of nitrofurantoin in human plasma by LC – MS / MS. Int J Pharm Sci Res 2017; 8(10): 4186-94.doi: 10.13040/IJPSR.0975-8232.8(10).4186-94.

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