Rapid, Sensitive and Simple LC-MS/MS Method Development and Validation for Estimation of Phenytoin in Human Plasma by using Deuterated Internal Standard

Dr. Ajay I. Patel¹, Kishna Ram*¹, Ms. Swati Guttikar², Dr. Amit Kumar J. Vyas¹,

Dr. Ashok B. Patel¹, Dr. Nilesh K. Patel¹, Dr. Vikash Trivedi²

¹B. K. Mody Government Pharmacy College, Rajkot, Gujrat (India)

²Veeda Clinical Research Pvt. Ltd., Ahmedabad, Gujrat (India)

*Corresponding Author E-mail: aipvk84@gmail.com, sundeshakishan555@gmail.com, swati.guttikar@veedacr.com, amitvyas77@gmail.com, abp2038@gmail.com, nkpatel99@gmail.com, vikas.trivedi@veedacr.com

ABSTRACT:

A rapid, sensitive, accurate and precise high-performance liquid chromatography–tandem mass spectrometric (HPLC– MS/MS) method equipped with Electro Spray Ionization (ESI) source, operating in the Positive ion and Multiple Reaction Monitoring (MRM) mode was developed and validated for the estimation of Phenytoin in human plasma using Phenytoin D₁₀ as an internal standard. Liquid-liquid extraction (LLE) technique was used to extract analyte and ISTDs from 0.2mL human plasma. The analytical separation was carried out in a reverse phase liquid chromatography by using C₁₈ (150 x 4.6mm, 5µm) column, and 2mM Ammonium Acetate in water (pH 6.3): Methanol (30:70% v/v) mobile phase at 1mL/min in isocratic mode. Analytes were monitored in multiple reactions monitoring (MRM) mode using the respective [M+H]⁺ ions, m/z 253.10→ 182.30  for Phenytoin and m/z 263.30 → 192.20 for the internal standard, respectively. The Phenytoin and Phenytoin D₁₀ retained at about 2.50 and 2.46 minutes respectively with total run time of 4 minute. The response of the LC-ESI-MS/MS method for both analyte and ISTD was linear over the dynamic range of 60-12000ng/mL with correlation coefficient r2 ≥ 0.9963. The Accuracy was well within the accepted limit of ± 20% at lower limit of quantification (LLOQ) and ± 15% at all the other concentrations in the linear range. Recovery of drug and ISTD was 87.52% and 86.42%, respectively. This method was fully validated for all the validation parameters and Stability studies. After optimization, the Bioanalytical method was validated according to USFDA, EMA and ANVISA guidelines for Bioanalysis.

KEYWORDS: Bioanalysis; LC-ESI-MS/MS; Biological fluids; Plasma; Liquid-Liquid Extraction (LLE); Bioanalytical guidelines.

INTRODUCTION:

Epilepsy a most common central nervous system disorder may cause persistent deformity and decreasing the quality of life, affecting 50 million people worldwide. Epilepsy is a chronic disorder that can influence human physiological, social and vocational functioning¹.

The term ‘epilepsy’ is used to define a group of disorders of the CNS characterized by paroxysmal cerebral dysrhythmia, manifesting as brief episodes (seizures) of loss or disturbance of consciousness, with or without characteristic body movements (convulsions). Sensory or psychiatric phenomena. These episodes are unpredictable and their frequency is highly variable^2,3.

Phenytoin (diphenylhydantoin), chemically is 5,5-diphenylimidazolidine-2,4-dione (Fig. 1), belongs to the category of antiepileptic also known as anticonvulsant, frequently prescribed for the treatment of all types of seizures except absence seizures and given orally in doses ranging from 200 to 600mg per day. When given orally, 70%–90% of the drug is absorbed and primarily binds to albumin with a binding of >90%. Due to its narrow therapeutic window of 10–20µg/mL, phenytoin monitoring is frequently required. Concentrations of >20 µg/mL are generally associated with toxic symptoms such as nystagmus, ataxia, and lethargy. The most common side effect of long-term use of phenytoin is gingival hyperplasia. The drug is extensively metabolized in the liver by first-order kinetics at the therapeutic concentrations. At higher concentrations, phenytoin elimination becomes dose dependent and zero order, thereby making therapeutic monitoring of the drug even more important^1,4.

The reported analytical methods used to determine phenytoin concentrations in biological samples includes Determination and Quantification of phenytoin in human plasma by liquid chromatography with electrospray ionization tandem mass spectrometry⁵, Determination of free level of phenytoin in human plasma by liquid chromatography/tandem mass spectrometry⁶, Validation of HPLC-MS method in positive ion mode for estimation of phenytoin in human plasma using phenytoin D₁₀ as internal standard¹, Rapid, sensitive and simple LC-MS/MS method development and validation for estimation of phenytoin in human plasma⁷, A simple isotope diluted electrospray ionization tandem mass spectrometry method for the determination of free phenytoin⁴, Saliva as a non-invasive biological sample to compare bioavailability of phenytoin formulation by LC-MS/MS⁸, A tandem mass spectrometry assay for the simultaneous determination of Acetaminophen, Caffeine, Phenytoin, Ranitidine and Theophylline in small volume paediatric plasma specimens⁹, Development and validation of an LC-MS/MS and comparison with GC-MS method to measure phenytoin in human brain dialysate, blood, and saliva¹⁰, Simultaneous determination of phenytoin and lamotrigine in human plasma using hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry¹¹, Simultaneous determination of phenytoin, carbamazepine and 10,11-carbamazepine epoxide in human plasma by high performance liquid chromatography with ultraviolet detection¹²,  Simultaneous rapid HPLC determination of Phenytoin and its prodrug Fosphenytoin in human plasma and ultra filtrate¹³, A novel HPLC method for determination of phenytoin in human plasma¹⁴, Determination of Phenytoin in human plasma by molecular imprinted solid phase extraction¹⁵, Determination of phenytoin in human plasma by a validated liquid chromatography method and its application to a bioequivalence study¹⁶.

Reported literature approaches for this analysis include most of methods have not use deuterated internal standard. According to EMEA guideline¹⁷ its uses in bioanalysis is preferable to minimise matrix interferences and this all methods were not validated according to USFDA, EMEA and ENVISA guidelines. The objective of the current analytical work was to ascertain a rapid, sensitive and simple LC–MS/MS method where deuterated internal standard was use; this enables to determine phenytoin in human plasma samples with good recovery and minimising matrix interferences. The main advantages of LCMS/MS include low detection limits and the ability to generate structural information of compounds^[18].The developed analytical method should incorporate a therapeutic range of free phenytoin concentration ranging from 60- 12000 ng/mL and method has to be established by validating according to EMEA, US-FDA and NVISA guidelines^17,19,20. The resulting analytical method serves as a prerequisite for further pharmacokinetic studies and ANDA filings in US countries as well as European countries.

Fig. 1 Chemical Structure of (A) Phenytoin Sodium, (B) Phenytoin D₁₀ (ISTD)

2. MATERIAL AND METHODS:

2.1 Chemicals and standards:

Phenytoin sodium (99.83%) and phenytoin D₁₀ (99.08%) were purchased from Simson pharma limited (Mumbai, India). Ammonium acetate, Methanol and Diethyl ether were purchased from Rankem, Biosolve and SD Fine Chem respectively. In all process Ultrapure water was used which are prepared in our laboratory using a Milli-Q Synthesis system. All chemicals were purchased in the highest purity available (AR/HPLC/MS grade).

2.2 Instrumentation and Optimized Experimental Conditions:

The API 3200 LC-MS/MS system where HPLC equipped with fully integrated triple quadrupole mass spectrometer (MDS/AB Sciex) and pulse counting discrete-dynode electron multiplier, with an electrospray ionization (ESI) interface operated in the positive mode and data were acquired using the Analyst software version 1.6.1 software. Determination was carried out using multiple reaction monitoring mode (MRM).

In Mass tuning, source parameter and compound parameter were optimised by the infusion of aqueous solution of Phenytoin and phenytoin D₁₀ and optimised parameters were as follows: curtain gas 25 units, collision gas (CAD) 5.0 units, Ion spray voltage 5500.0 V, temperature 500ºC, ion source gas 1, 50 units, ion source gas 2, 50 units. Nitrogen gas was used for curtain gas, ion source gas 2 (vaporizing gas), and zero air was used for ion source gas 1 (nebulizing gas). Declustering Potential (DP) 55eV, Entrance Potential (EP) 10 eV, Collision entrance Potential (CEP) 15 eV, Collision Energy (CE) 35eV, Collision Cell Exit Potential (CXP) 15 eV and Dwell time 200 milliseconds for Phenytoin and phenytoin D₁₀ were quantitated using the total ion current (TIC) of the multiple reaction monitoring (MRM) transitions. MRMs of m/z 253.10 → 182.30 (Drug Phenytoin), m/z 263.30 → 192.20 (ISTD Phenytoin D₁₀.

The Chromatographic separation was achieved using pumps (LC-20AD Series) and autosampler of Shimadzu for sample injection. Several different types of reversed phase columns have been tested. Finally, the C₁₈ (150 x 4.6mm, 5µm) (Nacalai Tesque Inc.) was chosen in order to have sharp and symmetric peaks, correct retention times, and most importantly, a good intensity for drug and ISTD. For the mobile phase, 2mM ammonium acetate in water (v/v): methanol (30:70 %v/v) was optimised. An isocratic elution at a flow rate of 1.0 mL/min and injection volume was 10µL with a total run time of 4.0 min. Temperature for column and autosampler were kept 40±5ºC and 5±3ºC, respectively.

2.3 Preparation of Stock Solutions:

2.3.1 Phenytoin stock solution:

2.607mg of Phenytoin working standard was transferred to a 15mL tarson tube, dissolved in 1.164mL HPLC grade methanol to produce a solution of 2.00mg/mL. Corrected the above concentration of phenytoin solution accounting for its molecular weight (252.27g/mol), formula weight (274.25g/mol), potency (97.07%) and the actual amount weighed. The stock solution was stored in refrigerator at 2-8°C and used for maximum of 7 days. The stock solutions were diluted to suitable concentrations using methanol to obtain CC spiked solution and from these spiked solution, calibration curve (CC) standards, quality control (QC) samples and DIQC samples were prepared.

2.3.2 Phenytoin D10 stock solution (Internal standard): 0.968mg of Phenytoin D₁₀ ISTD was transferred to a 15mL tarson tube, dissolved in 0.950mL HPLC grade methanol to produce a solution of 1.00 mg/mL. Corrected the above concentration of phenytoin solution accounting for its molecular weight, formula weight, potency (98.08%) and the actual amount weighed. ISTD dilution (5000.0ng/mL) was prepared from ISTD stock solution. The stock solution was stored in refrigerator at 2-8°C and used for maximum of 7 days.

2.4 Calibration Curve Standards and Quality Control Samples:

Spiking solution SS ULOQ (600000.0ng/mL) was prepared from drug stock solution, and then serial dilution were perform to obtain up to SS LLOQ (3000.0 ng/mL) concentration. Same as QC spiking solution SS HQC (450000.0ng/mL) was prepared from drug stock solution, and then serial dilution were perform to obtain SS MQC-1 (225000.0ng/mL), SS MQC-2 (75000.0 ng/mL), SS LQC (9000.0ng/mL) and SS LLOQ QC (3000.0ng/mL) solutions.

Calibration curve standard consisting of a set of eight non-zero concentrations ranging from 60 to 12000ng mL/ml of phenytoin were prepared by spiking the respective spiking solution into blank plasma (2% spiking of drug into blank plasma) as per Table no.1. Prepared quality control samples consisted of concentrations of 60ng/mL (LLOQ QC), 180ng/mL (LQC), 1500ng/mL (MQC 2), 4500ng/mL (MQC 1) and 9000ng/mL (HQC) for phenytoin from respective spiking solution as per Table no. 2. These samples were stored at -78°C until use.

Table 1: Preparation of Calibration Curve Standard

Drug Conc. (ng/mL)	Volume Taken (mL)	Volume of Diluent (mL)	Total Volume (mL)	Spiking Sol. Conc. (ng/mL) Drug	Spiking Solution ID	Spiking Volume (mL)	Biological Matrix Volume (mL)	Total Volume (mL)	Spiked Conc. (ng/mL) Drug	STD ID
2000000	1.2	2.8	4.0	600000	SS STD1 or SS ULOQ	0.20	9.80	10.0	12000	STD1
600000	1.6	0.4	2.0	480000	SS STD2	0.20	9.80	10.0	9600	STD2
480000	1.0	1.0	2.0	240000	SS STD3	0.20	9.80	10.0	4800	STD3
240000	1.0	1.0	2.0	120000	SS STD4	0.20	9.80	10.0	2400	STD4
120000	1.0	1.0	2.0	60000	SS STD5	0.20	9.80	10.0	1200	STD5
60000	0.9	0.9	1.8	30000	SS STD6	0.20	9.80	10.0	600	STD6
30000	0.4	1.6	2.0	6000	SS STD7	0.20	9.80	10.0	120	STD7
6000	1.0	1.0	2.0	3000	SS STD8 or SS LLOQ	0.20	9.80	10.0	60	STD8

Table 2: preparation of calibration curve standard

Drug Conc. (µg/mL)	Volume Taken (mL)	Volume of Diluent (mL)	Total Volume (mL)	Spiking Sol. Conc. (ng/mL) Drug	Spiking Solution ID	Spiking Volume (mL)	Biological Matrix Volume (mL)	Total Volume (mL)	Spiked Conc. (ng/mL) Drug	QC ID
2000000	0.90	3.10	4.0	450000	SS HQC	0.20	9.80	10.0	9000	HQC
450000	1.60	1.60	3.2	225000	SS MQC1	0.20	9.80	10.0	4500	MQC1
225000	1.00	2.00	3.0	75000	SS MQC2	0.20	9.80	10.0	1500	MQC2
75000	0.96	7.04	8.0	9000	SS LQC	0.20	9.80	10.0	180	LQC
9000	1.20	2.40	3.6	3000	SS LLOQ QC	0.20	9.80	10.0	60	LLOQ QC

2.5 Sample Preparation:

In pre labelled tube, 0.200mL Aliquoted then 50µL of ISTD Dilution (5000.000ng/mL) were added to all the samples except STD Blank and 50µL of Methanol was added into STD BL sample and vortex to mix it. Then 50 µL of Extraction Buffer (50mM Ammonium Acetate in Water, w/v) were added to all the samples and vortex to mix it. After that 2.5mL of Diethyl Ether were added, cap the tubes and extract on extractor at 50rpm for 20 minutes.

Samples were Centrifuged at 4000 rpm at 10 ± 2°C for 05 minutes. About 2.0mL of supernatant were transfer in pre labelled tubes and evaporate the samples to dryness under nitrogen at about 40 ± 5°C. Dried samples were reconstituted with 400µL of Mobile Phase and vortex to mix. Then samples were transfer into pre labelled auto sampler vials, arrange them in the auto sampler and inject by using LC-ESI-MS/MS.

2.6 Method Validation:

The method was validated according to the United States Food and Drug Administration (US FDA)¹⁹, European Medicines Agency (EMEA)¹⁷ and Brazilian NVISA²⁰ guidelines for bioanalytical method validation. Method validation of phenytoin in human plasma were carried out for specificity, linearity, precision and accuracy, recovery, matrix effect, dilution integrity, Ruggedness and stability according to above mention guidelines.

2.6.1 Specificity:

The Specificity of the method was established by screening the standard blank plasma (without spiking with drug or internal standard). Specificity was performed by using 9 different plasma lots among which 6 lots of normal plasma having K₃EDTA as anticoagulant, 1 lot of haemolysed plasma, 1 lot of lypidemic plasma and 1 lot of plasma having sodium heparin as anticoagulant. Sample processed: STD BL and LLOQ and Two sets of CC and QC samples.

2.6.2 Linearity:

The linearity of the method was determined by using accepted standard plots associated with 8 points standard curve including at least one LLOQ and one ULOQ. Concentration of CC standards were calculate against the CC; and the linearity of the method was evaluated by ensuring the acceptance of precision and accuracy of CC standards. The regression (r² value) should be ³0.98.

2.6.3 Precision and Accuracy (P and A):

The precision of the analytes was measured by the percent coefficient of variation over the concentration range of LLOQ, low, middle-1, middle-2 and high quality control samples during the course of validation. The accuracy of the analytes was measured by calculating the mean values of the LLOQ, low, middle-1, middle-2 and high quality control samples to their respective nominal values, expressed as percentage. The between run precision and accuracy were established by using all the replicate samples at LLOQ, low, middle-1, middle-2 and high quality control samples for the three consecutive precision and accuracy batches.

2.6.4 Recovery:

Recovery is a measure of efficiency at which an analytical method recovers the analyte through sample processing step. The % mean recovery was determined by comparing the mean peak area of the 6 replicates of extracted plasma quality control samples at high, middle-1 and Low concentrations against respective mean peak area of the 6 replicates of un-extracted quality control samples at high, middle-1 and low concentrations.

2.6.5 Matrix Effect:

Matrix effect was determined by calculating the matrix factor. Matrix factor is ratio of analyte response in presence of matrix ions to in absence of matrix ions. Processed ten different lots of previously screened biological matrix of same anticoagulant (including two hemolytic and two lipidemic lot) and after extraction spike the extract with analyte at concentration equivalent to those in the low, and high QC extracted samples (n=1 at each level for each lot of biological matrix) and internal standard at its working concentration. Aqueous vials of neat solutions of analyte and internal standard with the concentrations equivalent to those in low and high QC extracted samples (n= 6 at each level) were prepared. % CV for ISTD normalized matrix factor at each level (high, medium, low) were calculate. The variability in these responses, expressed as CV %, was considered as the measure of relative matrix effect.

2.6.6 Dilution Integrity:

The dilution integrity experiment was intended to validate the dilution test to be carried out on higher phenytoin concentrations (above HQC), which may be encountered during real subject samples analysis. It is perform to demonstrate that dilution of samples should not affect P and A. Spike 2% of DISS solution (3.00mg/mL) with blank plasma to obtain AUL QC (60000.00ng/ml). AUL QC further 10 times diluted with plasma and analysed with 3 precision and accuracy batch.

2.6.7 Stability:

Stability experiments were performed to evaluate the analyte stability in stocks solutions and in plasma samples under different conditions, which may occur during sample analysis. Stock solution stability was perform by comparing area response of stability samples of analytes and the internal standard with the area response of sample prepared from fresh stock solutions. Short term stock solution stability (STSS), Long term stock solution stability (LTSS), Short term working solution stability (STWSS), Long term working solution stability (LTWSS), Freeze thaw stability, Benchtop stability, stability of extract, dry extract stability, freeze–thaw stability and Stability of analyte in blood (SAB) were performed at LQC and HQC level using six replicates at each level.

2.7 Ruggedness:

Ruggedness is perform to check any changes made within instrument laboratory and analyst does not affect precision and accuracy of method. For ruggedness two precision and accuracy batch processed and inject into different instrument and on same instrument with different column.

3. RESULTS AND DISCUSSION:

3.1 Method development:

In previous studies, most of methods have not use deuterated internal standard^5-9,11 and other method have low linearity range^1,4,10, but in these available studies one method is excellent but this have also drawback of retention of compound and did not perform all validation parameter and stability studies¹. In other reported method, determination of phenytoin in human plasma were carried out by HPLC-UV method^12-16 which having low sensitive then LC-MS/MS determination. In our LC-MS/MS method deuterated internal standard was use to minimise matrix interference and method is developed for broad linearity range to detect phenytoin present in human plasma with dynamic concentration range which cover almost 50 to 300mg phenytoin dose and validation is perform as per USFDA, EMEA and NVISA guidelines.

Drug and internal standard were first characterized by MS and MS/MS connected with flow injection analysis to ascertain their parent ions and to select daughter ion for use in selective-ion monitoring (SIM) mode. Positive ionization mode is selected to obtain M+1 peak of parent ion. Full scan spectra for phenytoin produced a predominant peak of (M+H) at m/z 253.10. Collisionally induced dissociation of the (M+H) ion produced more intense daughter fragment at m/z 182.30. The m/z 182.30 ion was optimized as the selected product ion of the precursor at m/z 253.10 for selected-reaction monitoring mass spectrometry. Using similar procedures parent and daughter ion was obtain at m/z 263.3 and m/z 192.2, respectively for internal standard.

LLE extraction technique is simple, rapid and has relatively less cost per sample²¹. LLE technique give clean extracts of drug and ISTD from human plasma by 50 mM ammonium acetate as extraction buffer and diethyl ether as extraction solvent. The chromatogram from LLOQ, ULOQ and blank plasma samples were shown in Fig. 2.  Acquire chromatograms using the Analyst software supplied by the LC-ESI-MS/MS manufacturers.

The calibration curve (fitted by first-order y = ax + b, where a = slope, b = intercept, x = concentration and y = peak area ratio of Drug to ISTD is plotted as the peak area ratio of Drug to ISTD on Y–axis vs. the nominal concentration of drug(s) on X– axis and WATSON LIMS software for the calculation. The concentration of the unknown samples is calculated using linear regression equation with a validated 1/x² weighting factor.

Fig. 2 Chromatogram of blank plasma, ULOQ and LLOQ samples

3.2 Method Validation:

3.2.1 Specificity:

Specificity was performed to check % Interference at Rt of Analyte and ISTD. The obtained % interference at Rt of analyte was <20% and at Rt of ISTD was <5% of respective LLOQ sample, so all 9 lots of plasma were pass as per acceptance criteria.

3.2.2 Linearity:

The calibration plots constructed during the time of validation were observed linear for the regular concentration range of 60-12000ng/mL for phenytoin with the correlation coefficient (r²) 0.9970.

3.2.3 Precision and Accuracy:

 Intra-run precision for LLOQ QC, LQC, MQC 2, MQC 1 and HQC ranged from 1.80-3.85, 1.85-4.29, 1.38-4.17, 1.76-2.86 and 2.33-3.65%, respectively. Inter-run precision for LLOQ QC, LQC, MQC2, MQC 1 and HQC was 6.43, 0.58, 3.614.72, 2.44 and 4.10% respectively.

% Mean Accuracy of all QC standards were found to be within ± 15 % for Intra-run accuracy. Inter-run mean accuracy for LLOQ QC, LQC, MQC2, MQC1 and HQC was 99.94, 92.96, 92.89, 96.51 and 95.25% respectively.

3.2.4 Recovery:

The % overall recovery with correction factor for drug and ISTD was found to be 87.52% and 86.42, respectively. The % CV for drug and ISTD was found to be 2.72 and 1.75, respectively.

3.2.5 Matrix Effect: % CV of ISTD Normalised Matrix Factor at HQC and LQC level was found to be 2.04 and 2.57, respectively.

3.2.6 Dilution Integrity:

DI experiment was carried out using 6 consecutive injection of DQC sample having concentration of 6000.00 ng/mL. % Mean accuracy and % CV was found to be 2.66 and 88.41, respectively.

3.2.7 Stability:

Using the mean of three replicates (n = 3), short term stock solution stability (STSS), long term stock solution stability (LTSS), short term working solution stability (STWSS) and long term working solution stability (LTWSS) was carried out. % Mean stability for drug and ISTD was with in acceptance criteria of ± 10%. Using the mean of six replicates (n = 6), bench top stability, stability of extract, dry extract stability, freeze thaw stability, and stability of analyte in blood was assessed for phenytoin and internal standard. The % CV and % mean stability was with in acceptance criteria show in Table-3.

Table-3: Stability Summary of Phenytoin

Stability	QC	Mean ± SD (ng/mL)	Accuracy/ stability	Precision (%) or (% CV)
Bench Top Stability (6 hours)	LQC HQC	173.61 ± 3.02 8928.27 ± 236.86	96.45 99.20	1.74 2.65
Stability of Extract (At ambient temp.) (2 hours) Stability of Extract (At 5 ± 3°C temp.) (24 hours)	LQC HQC LQC HQC	160.81 ± 4.04 8107.39 ± 223.56 184.11 ± 6.22 9834.08 ± 373.61	89.34 90.08 102.28 109.27	2.51 2.76 3.38 3.80
Dry Extract Stability (2 hours)  (-20 ± 5ºC)	LQC HQC	167.52 ± 6.25 8468.61 ± 244.21	93.07 94.10	3.73 2.88
Freeze Thaw Stability (5 Cycle)  (-20 ± 5 °C)  (-78 ± 8 °C)	LQC HQC LQC HQC	177.29 ± 12.35 8465.91 ± 191.88 176.96 ± 8.16 8476.34 ± 576.54	98.49 94.07 98.31 94.18	6.96 2.27 4.61 6.80
Stability of Analyte in Blood  (At ambient temp.) 0 hours 30 min. 2 hours. 0 hours 30 min. 2 hours	LQC LQC LQC HQC HQC HQC	0.1165 ± 0.002 0.1103 ± 0.005 0.1093 ± 0.002 5.867 ± 0.132 5.685 ± 0.122 5.515 ± 0.103	- 94.71 93.83 - 96.90 94.01	1.30 4.57 1.49 2.25 2.15 1.87
Stability of Analyte in Blood (At Ice bath temp.) 0 hours 30 min. 2 hours 0 hours 30 min. 2 hours	LQC LQC LQC HQC HQC HQC	0.1105 ± 4.46 0.1098 ± 2.33 0.1078 ± 2.78 5.545 ± 2.34 5.677 ± 1.60 1.60 ± 2.11	- 99.40 97.59 - 102.37 102.16	0.109 0.109 0.105 5.56 5.56 5.61

Table-4: Ruggedness Precision and Accuracy

Different Equipment and Different column				Different analyst
Sample	Mean conc. found ±SD (ng/mL) (n=6)	CV (%)	Mean accuracy (%)	Mean conc. found ±SD (ng/mL) (n=6)	CV (%)	Mean accuracy (%)
LLOQ QC	66.16±8.17	12.35	110.27	62.55±1.14	1.82	104.24
LQC	169.76±5.07	2.99	94.31	168.34±3.11	1.85	93.52
MQC 2	1389.05±25.24	1.82	92.60	1361.21±56.71	4.17	90.75
MQC 1	4222.75±33.93	0.80	93.84	4397.18±115.99	2.64	97.72
HQC	8774.05±84.35	0.96	97.49	8756.70±319.62	3.65	97.30

3.2.8 Ruggedness:

Ruggedness was performed by analysing previously passed P and A batch with different column and different analyst. The results from quality control samples are presented in Table-4.

4. CONCLUSION:

In conclusion, the method was developed and validated for the quantitation of phenytoin in stabilized human plasma over the concentration range of 60-12000 ng/mL using Phenytoin D₁₀ as internal standard. The precision and mean accuracy are within the acceptable limits. Constant recoveries were practical for HQC, MQC and LQC level. The stability tests were carried out throughout the line of validation reveals that the phenytoin was stable at different settings in plasma/blood samples. Finally, the method was also proved to be rugged by different equipment and Different Column and different analyst. Thus, method can be useful for quantitation of phenytoin in human plasma.

5. ACKNOWLEDGEMENTS:

Author is very thankful to Veeda clinical research Pvt. Ltd., Ahmedabad who provided great platform for work in bioanalytical division.

6. CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 19.05.2020           Modified on 15.07.2020

Accepted on 10.08.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(6):2937-2944.

DOI: 10.52711/0974-360X.2021.00515