Int J Pharm Pharm Sci, Vol 8, Issue 9, 61-70Original Article


NEW METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF FEBUXOSTAT IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY

NAROTTAM PAL1*, AVANAPU SRINIVASA RAO1, PIGILLI RAVIKUMAR2

1Department of Pharmaceutical Analysis, Bhaskar Pharmacy College, Yenkapally, Moinabad, Hyderabad, India, 50075, 2Bio-Analytical Department, Aizant Drug Research Solution Pvt. Ltd, Hyderabad, India, 50014
Email: narottampal8224@gmail.com

Received: 01 Apr 2016 Revised and Accepted: 22 Jul 2016

ABSTRACT

Objective: To develop a new method and validate the same for the determination of Febuxostat (FBS) in human plasma by liquid chromatography–mass spectrometry (LCMS).

Methods: The present method utilized reversed-phase high-performance liquid chromatography with tandem mass spectroscopy. Febuxostat D9 (FBS D9) was used as internal standard (IS). The analyte and internal standard were separated from human plasma by using solid phase extraction method. Zorbax Eclipse XDB, C8, 100 mm x 4.6 mm, 3.5 µm column was used and HPLC grade acetonitrile, 5 millimolar (mM) ammonium format (80: 20, v/v) as mobile phase, detected by mass spectrometry operating in positive ion and multiple reaction monitoring modes.

Results: The parent and production transitions for FBS and internal standard were at m/z 317.1→261.0 and 326.1→262.0 respectively. The method was validated for system suitability, specificity, carryover effect, linearity, precision, accuracy, matrix effect, sensitivity and stability. The linearity range was from 20.131 ng/ml to10015. 534 ng/ml with a correlation coefficient of 0.999. Precision results (%CV) across six quality control samples were within the limit. The percentage recovery of FBS and internal standard from matrix samples was found to be 76.57% and 75.03% respectively.

Conclusion: Present study describes new LC-MS method for the quantification of FBS in a pharmaceutical formulation. According to validation results, it was found to be a simple, sensitive, accurate and precise method and also free from any kind of interference. Therefore the proposed analytical method can be used for routine analysis for the estimation of FBS in its formulation.

Keywords: Febuxostat, Febuxostat D9, Liquid chromatography, Mass spectrometry

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/)
DOI: http://dx.doi.org/10.22159/ijpps.2016v8i9.11968

INTRODUCTION

Chronic hyperuricemia and gouty arthritis [1-4] have ailed humans for centuries. Recent advancement of research in the understanding of the mechanism of their progress has changed our perception of the disease process. In spite of these developments, the treatment options are limited. The FDA approval of FBS for the treatment [5-8] of gouty arthritis or hyperuricemia has been a significant step forward. Since its approval during 2009, FBS has been proved to be a safe and efficacious treatment, although concerns remain about its long-term effects and superiority over other anti-gout agents, such as allopurinol.

Chemically the compound FBS is known as 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-

1, 3-thiazole-5-carboxylic acid. Fig. 1 represents the chemical structure.

Fig. 1: Chemical structure of FBS

The literature survey indicates that there are certain methods available for the determination of FBS in bulk drug form, formulation, metabolites and in the spiked plasma sample. Spectrophotometric methods were developed [9, 10] for determination of FBS in bulk and formulation. Reverse phase liquid chromatography was developed [11-16] determination of FBS in bulk, formulation and plasma sample. One method was reported [17] for the determination of FBS by using ultra-pressure liquid chromatography/mass spectroscopy and also there were two methods [18, 19] which used liquid chromatography-mass spectroscopy. Our developed new method has got unique advantages over these existing methods which are as follows

The proposed LC-MS method is a simple and selective for the determination of FBS in human plasma. The method employs only 100 microliter of human plasma volume and achieved good sensitivity. Use of low plasma volume for the analysis, the sample to be collected per time point from an individual during the study is reduced significantly. This allows the inclusion of additional points. The analyte and the IS were extracted from plasma using one-step solid–phase extraction with no drying, evaporation and reconstitution steps. Solid–phase extraction allows higher recoveries and the elimination of possible interference from endogenous and exogenous components. Isotope-labeled compound used as an IS to get better precision and accuracy. The total run time (2.0 min) is short enough compared to existing methods and makes it an attractive bioanalytical procedure of FBS for bioavailability and also will be in pharmacokinetic studies which will be studied shortly.

MATERIALS AND METHODS

Instrumentation

HPLC: Shimadzu

Mass spectrophotometer: API 3000 LC-MS/MS system

Reagents

Reagents/materials-Manufacturer/supplier

Methanol-JT Baker

Acetonitrile (HPLC grade)-JT Baker

Ammonium formate (AR grade)-Merck

Formic acid (AR grade)-Merck

HPLC grade water-Rankem

Strata X polymeric sorbent cartridges (30 mg/1cc)-Phenomenex

Working standards

Drug: Febuxostat

Batch No: VL/S-FB-004/b

Purity: 99.59% (HPLC)

Supplier: Vivan life sciences private limited.

Drug: Febuxostat D9

Batch No: VL/D-FB-206/a

Purity: 99.96% (HPLC)

Supplier: Vivan life sciences private limited.

Mass spectrometry operating conditions

Compound Febuxostat Febuxostat D9
Detection Positive Positive
M/z 317.10 (parent) 326.10 (parent)
261.00 (product) 262.00 (product)
Ion spray voltage (ISV) 4000 V, 4000 V
Temperature (TEM °C) 550 °C 550 °C
Curtain gas (CUR) 10 psi 10 psi
Collision gas (CAD) 10 psi 10 psi
NEB 6 psi 6 psi
Declustering potential (DP) 26 V 26 V
Collision energy (CE) 26 V 26 V
Collision cell exit potential
(CXP) 12 V 12 V
Focusing potential (FP) 128 V 128 V
Entrance potential (EP) 10 V 10 V
Dwell time 200 ms 200 ms

Preparation of standard stock and plasma samples

FBS stock solution: Weighed about 10.00 mg of FBS working standard and transferred to a 5 ml clean glass volumetric flask, dissolved in HPLC grade methanol and made up the volume with the same to produce a solution of 2 mg/ml. Corrected the above concentration of FBS solution accounting for its potency and the actual amount weighed.

The stock solutions were diluted to suitable concentrations using a mixture of acetonitrile and HPLC grade water in the ratio of (60:40 v/v) for spiking into the plasma to obtain calibration curve (CC) standards, quality control (QC) samples and DIQC samples. For the preparation of calibration curve standards and quality control samples, two separate stock solutions were prepared and used. All other final dilutions (system suitability dilutions, aqueous mixture, etc.) were prepared in the mobile phase. FBS D9 Stock Solution (Internal standard): Weighed about 2.0000 mg of FBS D9 hydrochloride, transferred to a 2 ml volumetric flask, dissolved in HPLC grade methanol and made up the volume with the same to produce a solution of 1 mg/ml. Corrected the above concentration of FBS D9 accounting for its molecular weight, potency and the actual amount weighed. The stock solution was diluted to a suitable concentration using diluent for internal standard dilution.

Note: Stock solutions and further dilutions of FBS D9 were prepared under the yellow monochromatic light.

Biological matrix

Eight lots of K2-EDTA human plasma, including one lipemia and one hemolytic plasma, were screened for selectivity test. All eight human plasma lots, including hemolytic and lipemic plasma, were found free of any significant interference for FBS and IS.

Selectivity and matrix test was performed before bulk spiking. After bulk spiking, 300 µl aliquot of each of the spiked calibration standards and quality control samples were pipetted out into 5 ml RIA vial, and stored in a deep freezer at–70 °C, except twelve replicates each of LQC and HQC, which were stored in a deep freezer at–20 °C for generation of stability data at–20 °C.

Calibration curve standards and quality control samples

Calibration curve standard consisting of a set of ten non-zero concentrations ranging from 20.131 ng/ml to 10015.534 ng/ml of FBS were prepared. Prepared quality control samples consisted of concentrations of 20.799 ng/ml (LLOQ QC), 61.174 ng/ml (LQC), 1529.341 ng/ml (MQC1), 5097.802 ng/ml (MQC2) and 7608.659 ng/ml (HQC) for FBS as given in table 1 and table 2. These samples were stored at–70 °C until use. Twelve sets of LQC and HQC were stored at–20 °C deep freezer to check-20 °C stability. Twenty-four sets of quality control samples for dilution integrity were prepared by spiking about 1.60 times the higher standard concentration of FBS (16797.041 ng/ml). From these six sets each of two times dilutions and four times dilutions were performed.

Table 1: Calibration curve (standard)

Final concentration- CC ( ng/ml) Label
20.131 1
40.262 2
201.312 3
503.281 4
1006.561 5
2013.122 6
4026.245 7
6009.320 8
8012.427 9
10015.534 10

*CC- Calibration curve

Table 2: Quality control (sample)

LLOQ QC (ng/ml) 20.799
LQC 61.174
MQC1 1529.341
MQC2 5097.802
HQC 7608.659
D1QC (ULQC) 16797.041

*LLOQ- Lower limit of quantification, QC- Quality control, LQC- Low-quality control, MQC- Medium quality control, HQC-High quality control, DIQC- Dilution integrity quality controle. ULQC- Upper limit quality control.

Sample preparation

The samples were thawed at room temperature and vortexed to ensure complete mixing of the contents. 200 µl of the plasma sample was pipetted in 5 ml polypropylene RIA vial, 20 µl of internal standard dilution (30598.856 ng/ml of FBS D9) was added to it and vortexed, except in blank plasma samples where 20 µl diluents was added and vortexed. Then, 600 µl of 0.1% formic acid buffer was added and vortex. The analyte and the IS were extracted from plasma using one-step solid–phase extraction with no drying, evaporation and reconstitution steps. SPE allows higher recoveries and the elimination of possible interference from endogenous and exogenous components. The sample mixture was loaded into the strata X 33 µm polymeric sorbent (30 mg/1 ml) cartridges that were preconditioned with 1.0 ml of HPLC grade methanol followed by 1.0 ml Milli Q HPLC grade water (new cartridge for each sample). After applying the maximum pressure, the extraction cartridge was washed with 2 ml of Milli Q/HPLC grade water (1.0 ml of each time). Then the samples were eluted with 1 ml of mobile phase and transferred to autosampler loading vials (amber color) and loaded into the auto-sampler.

Method development

Chromatographic separation was achieved after several trials using various combinations of solvents like acetonitrile, methanol, buffer (ammonium acetate, ammonium formate, formic acid, acetic acid at different concentrations) with varying proportions of each component on different columns like C18 and C8 of different brands like Grace, Chromolith, Hypersil, Hypurity advance, Kromasil, Zorbax, Ace and Intertsil. Use of 5 mM ammonium formate buffer helped in achieving good response for the detection in the positive ionization mode.

Validation parameters

Chromatography: Chromatographic conditions were optimized to achieve good sensitivity and peak shapes for the compounds, as well as a runtime which could be as short as it is possible. The liquid chromatographic conditions were optimized after a number of trials. A mobile phase consisting of acetonitrile and ammonium formtate 5 mM buffer (80:20, v/v) was found the most suitable. Zorbax eclipse XDB, C8, 100 x 4.6 mm, 3.5 µm (make: Agilent technology) column was the most useful one for developing the method.

System suitability

System suitability study was performed by applying six consecutive applications of FBS and FBS D9 LLOQ concentration and thereby observing the results in terms of retention time and area response and calculating %CV.

Selectivity

Selectivity test of the developed new method was conducted by injecting blank human plasma, spiked six samples at concentrations of (ULOQ) for FBS and the internal standard in plasma, compared the responses FBS and internal standard in the blank with a mean response of applied (ULOQ). The peak area of FBS at the respective retention time in blank should not be more than 20% of the mean peak area of (ULOQ) of FBS. Similarly, the peak area of FBS D9 at the respective retention time in blank should not be more than 5% of the mean peak area of (ULOQ) of FBS D9. Observations were there, whether any interfering compounds appeared in the chromatogram.

Sensitivity

The lower limit of reliable quantification for FBS in human plasma was set in the concentration of the LLOQ 20.131 ng/ml. The precision and accuracy for the analyte should be calculated and results in terms of accuracy and precision should be verified whether they are within the limit.

Matrix effect

To predict the variability of matrix effects in samples from individual subjects, matrix effect was quantified by determining the matrix factor, which was calculated as follows:

Matrix Factor = Peak response ratio in the presence of extracting matrix (post extracted)/Peak response ratio in aqueous standards.

Six lots of blank biological matrices were extracted each in triplicates and post spiked with the aqueous standard at the low, high QC level, and compared with aqueous standards of the same concentration. The overall precision of the matrix factor is expressed as a coefficient of variation (CV %) and %CV should be<15%.

Recovery

The extraction recovery of FBS and FBS D9 from human plasma was determined by analyzing quality control samples. Recovery at three concentrations (LQC, MQC2 and HQC) was determined by comparing peak areas obtained from the plasma sample and the standard solution spiked with the blank plasma residue.

Linearity

A regression equation with a weighting factor of 1/(concentration ratio) 2 of FBS to FBS D9 concentration was judged to get the best fit for the concentration--detector response relationship for FBS in plasma. The correlation coefficient (r2) value should be greater than 0.99 in the designed concentration range for FBS.

Precision and accuracy

The accuracy of the developed method is defined as the absolute value of the ratio of the calculated mean values of the LLOQ, low, middle and high-quality control samples to their respective nominal values, expressed in percentage. The precision of the developed method was measured by the percent coefficient of variation over the concentrations of LLOQ QC, LQC, MQC and HQC samples during the course of validation

Stability studies

Stability studies were performed by taking the sample passed through different physicochemical conditions like bench top, freeze-thaw, wet extract, dry extract, autosampler, dilution integrity, long term stability, etc.

RESULTS AND DISCUSSION

Method development

Optimization of liquid chromatography and mass spectrometry conditions

A detailed comparative study of the various published methods with the newly developed method is discussed in table 3. During method development while conducting trials, it was observed that increase in the proportion of buffer in mobile phase resulted in increased retention time, reduction in flow rate below 1 ml/minute also increased the retention time, similarly reduction of column temperature below 40 °C also contributes in prolongation of retention time. However, there was no substantial amount of variation in the number of theoretical plates or tailing factor. With the optimized method condition, we could able to limit the moderate run time that is 2 min, which is short enough compared to the existing method [18, 19] which used liquid chromatography-mass spectroscopy.

Moreover, if we look at the peak obtained in the case of Febuxostat D7 in the method developed by Babu Rao Chandu, Kanchanamala Kanala, Nagiat T Hwisa, Prakash Katakam, Mukkanti Khagga [19] it seems there were certain impurities along with the compound as the main peak was closely accompanied with additional peak. It may be an interference effect also. However, in our proposed method no such issues are associated.

Table 3: Comparative table of FEB

Method Solvent system Con. range Detection Reference
UV spectroscopy Methanol 0.2-15 µgm/ml 315 nm [9]
UV spectroscopy Zero order Methanol 2-30 µgm/ml 314 nm
First order 1-30 µgm/ml 293-336 nm
RP-HPLC Sodium acetate, ACN 0.1-200 µgm/ml 254 nm [11]
RP-HPLC Methanol, OPA 45.42-2559.64 ngm/ml 310 nm [12]
RP-HPLC Ammonium acetate, ACN 50-400 µgm/ml 275 nm [13]
RP-HPLC Methanol, ACN 40-100 µgm/ml 218 nm [14]
RP-HPLC Pot. di. H. PO4, ACN 5-60 µgm/ml 320 nm [15]
RP-HPLC Methanol, Sodium acetate 250-8000 ng/ml 315 nm [16]
UPLC-MS Formic acid, ACN 2-10000 µgm/ml MRM-MD [17]
LC-MS Formic acid, CAN, water 10-20000 ng/ml MRM-MD [18]
LC-MS Ammonium formate, ACN 1-8000 ng/ml MRM-MD [19]

*CAN-Acetonitrile, OPA-Ortho phosphoric acid, Pot. di. H. PO4-Potassium dihydrogen phosphate, MRM-Multi reaction monitoring mode, MD-Mass detector.

In the present method, separation was achieved by using mobile phase as Acetonitrile and 5 mM ammonium formate (80:20, v/v) in isocratic elution technique at a flow rate of 1.00 ml/min. fig. 2 represents the mass spectra of FEBU and FEBU D9. Fig. 3 represents chromatogram of the blank plasma and blank plasma with internal standards.

Method Validation

System suitability

Consecutive six injections were applied for FBS and IS LLOQ concentration in the chromatographic system. The mean retention time was found to be 0.9733 min and 0.9616 min respectively. If it is compared with any other existing method [18, 19] it is easy to understand the rapid nature of the present method. Mean area, standard deviation, and %CV were found to, 18528.0 and 2508497.3, 1805.26 and 223043.3, 9.74 and 8.89 for FBS and IS respectively which are well within the acceptable limit.

Selectivity and specificity

The selectivity and specificity of the present analytical method were established by examining any interfering compounds which elute along with FBS. The response of both analyte and IS in blanks was compared with the mean response of injected LLOQ.

Fig. 2: Q1/Q3 mass spectrum of FBS along with the IS


Fig. 3: Chromatogram of blank plasma sample of FBS and FBS D9

There were no interfering peaks formed at FBS retention time and IS retention time in the plasma blanks. Fig. 4 shows chromatograms of blank human plasma samples. The results were shown in table 4 and table 5. The analytical study of FBS and FBS D9 using the multiple reaction monitoring functions was highly selective and no interfering compounds were observed.

Recovery

As the sample and internal standard FBS D9 were extracted from human plasma, and the test for recovery was determined by analyzing quality control samples (LQC, MQC2 and HQC) by means of comparison of peak areas obtained from the plasma sample and the standard solution spiked with the blank plasma residue, the average percentage recoveries were found to be 76.56% and 75.03% respectively. This yield is up to the mark and comparable with any other mass spectrophotometric method. Detailed results are narrated in table 6 and table 7.

Table 4: Result of system suitability

S. No. tR in Minutes Peak response (area) LLOQ
Analyte Internal standard
1 0.97 0.96
2 0.97 0.96
3 0.97 0.96
4 0.97 0.96
5 0.98 0.97
6 0.98 0.96
Mean (±) 0.973 0.961
SD 0.00516 0.00408
CV% 0.530 0.424

*tR-Retention time, LLOQ-Lower limit of quantification

Table 5: Specificity of FBS and internal standard

Sample Id FBS Peak area IS peak area % interference at tR of FBS % interference at tR of IS
ULOQ FBS 1 6125002 0 NA 0
ULOQ FBS 2 5948204 0 NA 0
ULOQ FBS 3 5658120 0 NA 0
ULOQ FBS 4 5450620 0 NA 0
ULOQ FBS 5 5408965 0 NA 0
ULOQ FBS 6 6844515 0 NA 0
The mean response of FBS in presence of IS 5905904
Mean response of FBS D9 in presence of FBS 0 0
Blank+IS (FBS D9) 1 0 1996826 0 NA
Blank+IS (FBS D9) 2 0 2123014 0 NA
Blank+IS (FBS D9) 3 0 1858814 0 NA
Blank+IS (FBS D9) 4 0 2087960 0 NA
Blank+IS (FBS D9) 5 0 2133141 0 NA
Blank+IS (FBS D9) 6 0 1999478 0 NA
Mean response of FBS D9 in presence of FBS 2033210
Mean response of FBS 0

* ULOQ-Upper limit of quantification, FBS-Febuxostat, IS-Internal standard, tR-Retention time, NA-Not applicable.

Table 6: Recovery of FBS from matrix

Standard Identifying code Unextracted standard peak area Identifying code Extracted matrix standard peak area %recovery
LQC AQS-LQC-1
AQS-LQC-2
AQS-LQC-3
AQS-LQC-4
AQS-LQC-5
AQS-LQC-6

51867

53188

54426

52778

53780

54577

EXT-LQC-1

EXT-LQC-2

EXT-LQC-3

EXT-LQC-4

EXT-LQC-5

EXT-LQC-6

39587

39724

39027

38041

37193

37261
Mean (±)
SD
CV%		 534360.0
1035.04
1.94		 38472.2
1131.98
2.94		 72%
MQC

AQS-MQC-1
AQS-MQC-2

AQS-MQC-3

AQS-MQC-4

AQS-MQC-5

AQS-MQC-6

4120459

3997420

4263493

4340363

4040216

4280130

EXT-MQC-1 EXT-MQC-2

EXT-MQC-3

EXT-MQC-4

EXT-MQC-5

EXT-MQC-6

3214184

3067145

3161738

3051183

3068832

3026803
Mean (±)
SD
CV%

4173680.2

140637.05

3.37

3098314.2

72971.65

2.36

 74.23%
HQC

AQS-HQC-1

AQS-HQC-2

AQS-HQC-3

AQS-HQC-4

AQS-HQC-5

AQS-HQC-6

5731755

5649408

5618990

5728449

5696802

5765760

4859028

4793110

4777081

4717922

4654670

4736922
Mean (±)
SD
CV%

5698527.3

55247.60

0.97

4756455.5

70023.94

1.47

 83.47%

*LQC-Low quality control, AQS-Aqueous, EXT-Extracted, SD-Standard deviation, CV-Co-efficient of variance, MQC-Medium quality control, HQC-High Quality control.

Table 7: Recovery of internal standard from matrix

Standard Identifying code Unextracted peak area Identifying code Extracted peak area %recovery
Internal standard

AQS-LQC-1

AQS-LQC-2

AQS-LQC-3

AQS-LQC-4

AQS-LQC-5

AQS-LQC-6

AQS-MQC-1

AQS-MQC-2

AQS-MQC-3

AQS-MQC-4

AQS-MQC-5

AQS-MQC-6

AQS-HQC-1

AQS-HQC-2

AQS-HQC-3

AQS-HQC-4

AQS-HQC-5

AQS-HQC-6

2541331

2615551

2662715

2613782

2643611

2689044

2376143

2315155

2406262

2544363

2336335

2449825

2291491

2257716

2264027

2310835

2307111

2311513

SPIKED-LQC-1

SPIKED-LQC-2

SPIKED-LQC-3

SPIKED-LQC-4

SPIKED-LQC-5

SPIKED-LQC-6

SPIKED-MQC-1

SPIKED-MQC-2

SPIKED-MQC-3

SPIKED-MQC-4

SPIKED-MQC-5

SPIKED-MQC-6

SPIKED-HQC-1

SPIKED-HQC-2

SPIKED-HQC-3

SPIKED-HQC-4

SPIKED-HQC-5

SPIKED-HQC-6

1942265

1948802

1955042

1886018

1844533

1812939

1882419

1791023

1866519

1789813

1778645

1770304

1831302

1806206

1814292

1762670

1724347

1757634
Mean (±) 2440933 1831376.3
SD 154156.27 69079
%CV 6.32 3.77

*FBS-Febuxostat, LQC-Low quality control, AQS-Aqueous, SD-Standard deviation, CV-Co-efficient of variance, MQC-Medium quality control, HQC-High Quality control.

Linearity, accuracy, and precision

Linearity, accuracy, and precision: The calibration curve was constructed using 8 calibration standards ranging from 20.131ng/ml to 10015.534ng/ml. a straight line fit was made through the data points. The correlation coefficient was found to be ≥0.999. The lower limit of quantification (LLOQ) was found to be 20.799ng/ml. Accuracy was calculated in terms of percentage recovery and precision in terms of percentage coefficient variation. For the concentration of LLOQ the accuracy result and precision value were found to be 100.10% and CV% 5.69 respectively. The test result for inter-batch accuracy was 97.91%; precision for LLOQ, LQC, MQC and HQC was 4.96, 3.77, 3.47 and 2.18 respectively. The test result for inter-batch accuracy was 99.77% (first batch), 97.24% (second batch); precision for LLOQ, LQC, MQC and HQC for the first batch was 3.04, 3.62, 4.44 and 0.64; for second batch 4.49, 1.13, 2.90 and 0.69 respectively. The test result for intraday accuracy was 98.51%; precision for LLOQ, LQC, MQC1, MQC2 and HQC was 3.85, 2.72, 4.36, 4.46 and 1.49 respectively. All the results of linearity, accuracy, and precision were within the limit. Fig. 4 represents the calibration curve, fig. 5 to fig. 9 represent the chromatograms of FBS and FBS D9 for the concentrations of LLOQ, LQC, MQC1 and HQC. Table 8 to table11 contains the results of accuracy and precision.

Fig. 4: Calibration curve for regression analysis of FBS


Fig. 5: Chromatogram of LLOQ QC sample of FBS and FBS D9


Fig. 6: Chromatogram of LQC sample of FBS and FBS D9


Table 8: Precision and accuracy for LLOQ (sensitivity)

FBS

 Identifying code

Nominal (ng/ml)

% Accuracy

LLOQ-1

19.892

98.81

LLOQ-2

20.982

104.23

LLOQ-3

18.861

93.69

LLOQ-4

19.183

95.29

LLOQ-5

21.931

108.98

LLOQ-6

20.054

99.62

Mean

20.1518

 

SD

1.14587

 

CV%

5.69

 

% nominal

100.10

 

*Number of replicates = 6, FBS-Febuxostat, LLOQ-Lower limit of quantification, SD-Standard deviation, CV-Co-efficient of variance.


Fig. 7: Chromatogram of MQC1 sample of FBS and FBS D9


Fig. 8: Chromatogram of MQC2 sample of FBS and FBS D9


Table 9: Inter batch precision and accuracy

Batch LLOQ (ng/ml) LQC (ng/ml) MQC1 (ng/ml) MQC2 (ng/ml) HQC (ng/ml)
QC 20.779 %Accuracy 61.174 %Accuracy 1529.341
Mean 20.3139 97.6 58.1238 95.01 1529.5868
SD 1.00710 2.18880 3.79
CV% 4.96 3.77 99.49

*Number of replicates = 30, LQC-Low quality control, MQC-Medium quality control, HQC-High Quality, SD-Standard deviation, CV-Co-efficient of variance control.


Fig. 9: Chromatogram of HQC sample of FBS and FBS D9


Table 10: Intraday precision and accuracy

LLOQ (ng/ml) LQC (ng/ml) MQC1 (ng/ml) MQC2 (ng/ml) HQC (ng/ml)
QC 20.799 %Accuracy 61.174 %Accuracy 1529.341
Mean 20.4502 98.32 58.2138 95.16 1493.5383
SD 0.78632 1.58526 65.06109
CV% 3.85 2.72 4.36

*Number of replicates = 12, QC-Quality control, LLOQ-Lower limit of quantification, LQC-Low quality control, MQC-Medium quality control, HQC-High Quality, SD-Standard deviation, CV-Co-efficient of variance control.

Table 11: Intra batch precision and accuracy

Batch LLOQ (ng/ml) LQC (ng/ml) MQC1 (ng/ml) MQC2 (ng/ml) HQC (ng/ml)
QC 20.799 %Accuracy 61.174 %Accuracy 1529.34
1st batch
Mean 20.6940 99.50 58.7045 95.96 1457.2373
SD 0.62943 2.12693 64.67799
CV% 3.04 3.62 4.44
N 6 6 6
2nd batch
Mean 20.2063 97.15 57.7230 94.36 1529.839
SD 0.90631 0.65334 44.34646
CV% 4.49 1.13 2.90

*N-Number of replicates = 6, QC-Quality control, LLOQ-Lower limit of quantification, LQC-Low quality control, MQC-Medium quality control, HQC-High Quality control, SD-Standard deviation, CV-Co-efficient of variance.

Carry over effect

The sequence of injections consisting two blank samples and two samples of ULOQ concentration were analyzed alternately to find out if there is any carry over affect on the blank sample. It was found that there was no carryover effect observed in the present method.

Stability results

As stability studies were performed by taking FBS passed through different physicochemical conditions like bench top, freeze-thaw, wet extract, dry extract, autosampler, dilution integrity, long-term stability, the mean response, percentage recovery and percentage coefficient variation were found within the limit. Table 12 represents the results of stability studies in details.

Dilution integrity

Dilution integrity was performed by taking two times and four times dilution of ULOQ concentration (16797.041 ng/ml). The percentage accuracy and %CV were found to be within the acceptance criteria (table 13).

Table 12: Stability data of QC samples in human plasma

Nominal Conc Stability Mean (ng/ml) % Accuracy Precision (CV %)

LQC

(61.174 ng/ml)

 Bench top 15 h 59.1428 96.68 5.90
Freeze-thaw 59.7635 97.69 3.64
Wet extract 62.6802 102.46 5.75
Autosampler 60.0737 98.20 4.16
Freshly spiked QC 58.4463 95.56 1.77
60 d 59.4030 95.56 0.72

HQC

(7608.946 ng/ml)

 Bench top 15 h 7608.659 100.95 1.20
Freeze-thaw 7737.6602 101.70 1.61
Wet extract 7803.0808 102.56 1.19
Autosampler 7714.5567 101.39 1.22
Freshly spiked QC 7756.0290 101.96 0.72
60 d 7656.8233 100.63 0.80

*Number of replicates, QC-Quality control, LQC-Low quality control, HQC-High quality control, CV-Co-efficient of variance.

Table 13: Data acquired on dilution integrity

Nominal concentration (NC) ng/ml

DIQC

Two times dilution

Four times dilution

NC = 16797.041

% Accuracy

NC = 16797.041

% Accuracy

Mean

16355.7257

97.37

16542.7382

98.49

SD

444.99782

 

436.31186

 

CV%

2.72

 

2.64

 

*DIQC-Dilution integrity quality control, NC-Nominal concentration, SD-Standard deviation, CV-Co-efficient of variation.

CONCLUSION

The new developed method is a rapid, simple, specific and accurate liquid chromatography mass spectrophotometry for the determination of FBS as the results of all the validation parameters were found within the limit. The sophisticated solid phase extraction technique has yielded in high precision values. As per Cmax of FBS the range was designed and the method was very sensitive with a low value of LLOQ. The newly developed method can be used for regular determination of Febuxostat in the laboratory.

CONFLICT OF INTERESTS

Declared none

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