Quantification of Busulfan in Pharmaceutical Formulations by Analytical HPLC

GRD Journals- Global Research and Development Journal for Engineering | Volume 6 | Issue 3 | February 2021 ISSN- 2455-5703

Quantification of Busulfan in Pharmaceutical Formulations by Analytical HPLC Ch. Venkata Kishore Assistant Professor Department of Chemistry Dr. L.B. College, Visakhapatnam

V. Tejeswara Rao Assistant Professor Department of Chemistry MVR College, Visakhapatnam, Andhra Pradesh 530045

K. Balaji Assistant Professor Department of Chemistry MVR College, Visakhapatnam, Andhra Pradesh 530045

K. Raghu Babu Professor Department of Chemistry Andhra University Engineering College(A), Visakhapatnam

Abstract The present study was conducted to validate an analytical procedure for the Quantification of Busulfan in Pharmaceutical Formulations. The analytical test attribute Busulfan was evaluated as per the guidelines of ICH Q2 (R1). It is a new simple, accurate, precise and reproducible HPLC method has been developed for the estimation of Busulfan (1,4-butanediol dimethanesulfonate) in its inject able dosage. Thus, the proposed HPLC method can be successfully applied for the routine quality control analysis of formulations. A mixture water, acetonitrile and tetrahydrofuran at 30:65:5 (V/V/V) ratios were prepared and used as mobile phase.

Keywords- Busulfan, HPLC, ICH Q2 (R1), Validating the Assay

I. INTRODUCTION When Busulfan hydrolyses in aqueous media, the methanesulphonate groups are released. The half-life of the intermediate, 4methanesulphonyloxybutanol, is extremely short, which makes it unlikely that it is jointly responsible for the biological action of Busulfan [1]. Eventually, as high-dose busulfan emerged as an important component of preparative regimens in the early 2000s, intravenous formulations were marketed to overcome the disadvantages of the original oral compound’s bioavailability [2].

Nonetheless, studies comparing intravenous versus oral administration of busulfan used identical dosing frequencies (again, typically q6 h) in their protocols to avoid confounding variables [3].

The second study was done in pediatrics, and found that the rate of veno-occlusive disease (VOD) was higher in the q24 h group compared to the q6 h group [4]. Not only does the reduced dosing frequency yield direct drug cost savings, but it also decreases pharmacy resources required to prepare the IV admixture from four times daily, to just once per day. In particular, busulfan’s stability is only 12 hours once admixed, so daily administration is more practical for pharmacy [5].

This drug used in study of platelet-transported serotonin in liver reconstruction [6]. Tonicity which includes interstitial "busulfan lung", hyper pigmentation, seizures, veno-occlusive disease, emesis, and wasting syndrome [7-8]. Recently, intravenous Busulfan formulations were introduced on to the market, in order to minimize variations of inter- and intra-patient systemic exposure, and to provide complete dose assurance [9-11].

ICH- international council for harmonization of technical requirements for pharmaceuticals for human use (ICH) is unique in bringing together the regulatory authorities and pharmaceutical industry to discuss scientific and technical aspects of drug registration.Q2 (R1) Validation of analytical procedures of methodology is document presents a discussion of the characteristics for consideration during the validation of the analytical procedures included as part of registration applications submitted within the EC, Japan and USA. This document does not necessarily seek to cover the testing that may be required for registration in, or export to, other areas of the world. Furthermore, this text presentation serves as a collection of terms, and their definitions, and is not intended to provide direction on how to accomplish validation. These terms and definitions are meant to bridge the differences that often exist between various compendia and regulators of the EC, Japan and USA. The objective of the analytical procedure should be clearly understood since this will govern the validation characteristics which need to be evaluated. Typical validation characteristics which should be considered are Accuracy, Precision, Repeatability, Intermediate Precision, Specificity, Detection Limit, Quantization Limit, Linearity, Range[12-13].

II. TEST SYSTEM – –

Instrument Name: High performance liquid chromatography Make: Shimadzu All rights reserved by www.grdjournals.com

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Quantification of Busulfan in Pharmaceutical Formulations by Analytical HPLC (GRDJE/ Volume 6 / Issue 3 / 004)

– –

Model: LC-2030C Software: LC Solutions

III. EQUIPMENT’S Following equipment’s were used for the study.

S. No. 1 2 3 4 5

Equipment Weighing Balance High Performance Liquid Chromatography Ultrasonic cleaner Micropipette pH Meter

Model XS205 Dual Range LC-2030C 101/250 SL-1000 PICO +

Make/Supplier Mettler Toledo Shimadzu PCI Analytics Rainin Labindia

IV. CHEMICALS / CONSUMABLES S.No. 1 2 3 4

Name Acetonitrile Sodium diethyl dithiocarbomatetrihydrate N,N Dimethyl acetamide Tetrahydron

Grade High Performance Liquid Chromatography High Performance Liquid Chromatography High Performance Liquid Chromatography Analytical Regent Grade

Manufacturer Merck Limited Merck Limited Merck Limited Merck Limited

V. ANALYTICAL CONDITIONS – – – – – – – – – – – –

Instrument: Shimadzu Class VP Binary pump LC-10ATvp, SIL-10ADvp Auto sampler, CTO 10Avp Column Temperature Oven, SPD-10Avp UV-Visible Detector. All the components of the system are controlled using SCL-10Avp System Controller. Data acquisition was done using LC Solutions software.

Mobile phase: Water, acetonitrile and tetrahydrofuran at 30:65:5 (V/V) ratio.

Column: YMC Pack ODS-A (150 x 4.6) mm, 3µm Detection Wavelength: 280 nm Flow rate: 1.5 mL / min Injection volume: 20 µL Run time: 15 minutes Column temperature: 25° C Sample cooling rack: 20° C Diluent: Methanol Run Time: 12.0 Minutes Retention Time: 4.4 min (approximately)

VI. PROCEDURE A. Preparation of Mobile Phase Mobile phase is prepared by mixing water, acetonitrile and tetrahydrofuran at 30:65:5 (V/V) ratio and sonicated the resulting solution well, degassed it using vacuum filtration through 0.22 µm filter.

B. Preparation of Sodium Diethyl Dithiocarbomatetrihydrate Stock Solution (Derivatising Reagent) Weigh and transfer accurately 1000 mg of Sodium diethyl dithiocarbomatetrihydrateinto 25 mL volumetric flask, add 10ml of N, N Dimethyl acetamide sonicate to dissolve and make up to the volume with N, N Dimethyl acetamide and mix well.

C. Preparation of Derivatisation Blank Solution Transfer 5.0 mL of Sodium diethyl dithiocarbomatetrihydrate stock solution in to 25 mL volumetric flask, keep the solution in water bath at 60°C for 30mins and add 2 mL of diluent shake and keep solution in water bath at 60°C for 20mins,shake it occasionally. Retrieve the sample and allow it to cool for room temperature and make up to volume with diluent and filter through 0.22 µm PVDF Filter.

D. Preparation of Standard Stock Solution Weigh and transfer 30 mg of Busulfan standard to 50 mL volumetric flask, dissolve with25 mL of N, N Dimethyl acetamide and dilute to volume with diluent and mix well.

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Quantification of Busulfan in Pharmaceutical Formulations by Analytical HPLC (GRDJE/ Volume 6 / Issue 3 / 004)

E. Preparation of Derivatisation Standard Solution Transfer 5.0 mL of Sodium diethyldithiocarbamatetrihydrate stock solution in to 25 mL volumetric flask, keep the solution in water bath at 60°C for 30 min and add 2 mL of standard stock solution, shake and keep solution in water bath at 60°C for 20minutes, shake it occasionally. Retrieve the sample and allow it to cool for room temperature and make up volume with diluent and filter through 0.22 µm PVDF Filter.

F. Preparation of Test Sample Stock Solution Transfer 5.0 mL of test sample to 50 mL volumetric flask, rinse the pipette 2 times with diluent and 25ml of diluent sonicate for 10 minutes with occasional shaking after that vortex for 10 minutes and diluted to volume with diluent and mix well.

G. Preparation of Derivatisation Test Sample Solution Transfer 5.0 mL of Sodium diethyldithiocarbamatetrihydrate stock solution in to 25 mL volumetric flask, keep the solution in water bath at 60°C for 30 min, add 2 mL of test sample stock solution, shake and keep solution in water bath at 60°C for 20minutes, shake it occasionally. Retrieve the test sample and allow it to cool for room temperature, make up volume with diluent and filter through 0.22 µm PVDF Filter.

H. – – –

Suitability Solution The Tailing factor for Busulfan peak in standard solution should be NMT 2.0.

The relative standard deviation for Busulfan peak from five replicate injection of Standard solution should be NMT 2.0%.

The theoretical plates for Busulfan peak in standard solution should be NLT 2000.

VII.

CALCULATION

AT WS DT P 100 -------- x -------- x --------x -------- x--------AS DS V 100 LA AT: Average area of Busulfan peak obtained from chromatogram of Sample preparation AS: Average area of Busulfan peak obtained from chromatogram of Standard Preparation WS: Weight of Busulfan standard in mg DS: Dilution of standard preparation in mL DT: Dilution of sample preparation in mL P: Potency of Busulfan standard on as is basis V: Volume of sample taken in mL LA: Label amount of Busulfan in mg/mL % Assay =

VIII. METHOD VALIDATION The method for determination of Busulfan was validated in terms of precision (System precision and Method precision), Specificity (Interference, Forced degradation, Linearity), Intermediate Precision, Accuracy, Robustness, Stability of Analyte in solution, Filter compatibility and System Suitability of overall validation study.

A. Linearity The linearity of an analytical procedure is its ability to obtain test results which are directly proportional to the concentration of analyte in sample. The linearity of Busulfan is established by analyzing Linearity solutions of different concentrations from 50 % to 150 % of working concentration of method for Assay. The Linearity curve is plotted for area versus concentration (Figure 1).

The linearity results are tabulated in the below table 1. Chromatogram of Linearity at 100% level is exhibited below as figure 2.

Linearity Level 50 % 75 % 100 % 125 % 150 % Slope

Table 1: Results of Linearity Concentration (µg/mL) Area (Average) 27.1 1873386 36.9 2520268 49.2 3444590 61.5 4186867 73.8 4948738 Correlation coefficient (R) : 0.999 : 66205 Y-intercept bias at 100% level : 3.0

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Quantification of Busulfan in Pharmaceutical Formulations by Analytical HPLC (GRDJE/ Volume 6 / Issue 3 / 004)

Fig. 1: Linearity Graph

Fig. 2: Chromatogram of Linearity at 100% level

B. Stability of Analyte in Solution Stability of analyte in solution is evaluated for the standard and sample solutions. The standard and sample solutions are prepared and analyzed as per the analytical procedure. A portion of these solutions were preserved at room temperature and 2-8°C and analyzed at different time intervals from the time of preparations. The results are calculated from initial versus over a period of time. The results are tabulated in below table 2 and table 3.

Time Interval Initial 24 hours 48 hours Time Interval Initial 24 hours 48 hours

Table 2: Stability of Standard Solution %Assay of Busulfan % Difference Room Temperature 2-8°C Room Temperature 100 NA 100.3 100.3 -0.30 101.3 101.3 -1.30 Table 3: Stability of Sample Solution %Assay of Busulfan % Difference Room Temperature 2-8°C Room Temperature 100.5 NA 99.7 101.3 0.80 101.0 102.5 -0.50

2-8°C -0.30 -1.30

2-8°C -0.80 -2.0

C. Filter Compatibility A study to establish the stability of filter will be conducted by using two different types of filters 0.2 µm Nylon and 0.22µm PVDF filters. Standard and sample solutions were prepared and injected to establish the stability of filters. The results are tabulated in below table 4 and table 5.

Table 4: Compatibility for Standard Solution Busulfan standard Time Interval 0.2 µm Nylon 0.22µm PVDF filters Injection-1 99.9 99.9 Injection-2 100.2 99.9 Average 100.0 99.9

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Quantification of Busulfan in Pharmaceutical Formulations by Analytical HPLC (GRDJE/ Volume 6 / Issue 3 / 004)

Table 5: Compatibility for Sample Solution Busulfan standard Time Interval 0.2 µm Nylon 0.22µm PVDF filters Injection-1 101.3 100.5 Injection-2 101.4 100.5 Average 101.3 100.5

IX. RESULTS – OVERALL SUMMARY OF VALIDATION Validation Parameters

Precision

Specificity

Acceptance Criteria Error! Reference source not found.

The relative standard deviation for Busulfan peak area from five replicate injection of standard solution should be not more than 2.0% Error! Reference source not found.

The relative standard deviation of assay results obtained from six sample preparations should not be more than 2.0% Interference No Interference should be observed at the retention time of Busulfan peak in the chromatograms obtained from the diluent, Blank and placebo.

Validation Parameters

Linearity

Intermediate Precision

Accuracy

% RSD

Busulfan

0.3%

Busulfan

0.1%

There is no interference is observed at the retention time of Busulfan peak in the chromatogram obtained from the diluent, Blank and placebo.

Results

Drug Product (FP) % Purity Purity Array of Stress degradation Angle Threshold As Such (Unstressed) 0.681 19.281 Acid degradation 2.2 0.793 17.890 Alkali degradation 8.6 0.796 18.528 Oxidation degradation 2.2 1.069 66.090 UV degradation Not degraded 0.690 18.648 Thermal degradation 2.6 0.678 19.149 Neutral degradation 1.7 0.747 18.585 Busulfan Correlation coefficient (R) 0.999 slope of regression line 66205 Y-intercept of regression line 104381

Y-intercept bias at 100% level

Precision 0.1%

Recovery at each level and overall average recovery of assay results should be between 98.0% and 102.0%

3.0

Intermediate Precision 0.6%

0.4%

Acceptance Criteria –

Component name

Acceptance Criteria

Error! Reference source not found.

Calculate the % degradation against as such test preparation for each condition, in any of one condition degradation should be achieved between 5.0% to 20.0%.

Each degradation sample, purity angle should be less than the purity threshold for Busulfan peak.

– Correlation coefficient should not be less than 0.999 for Busulfan.

– Report the slope of regression line.

– Report the Y-intercept of regression line.

– Y-intercept bias at 100 % level should be between ± 5.0 % for Busulfan.

– The relative standard deviation results obtained from six sample preparations should not be more than 2.0% – The cumulative %RSD of method precision and intermediate precision results obtained from twelve sample preparations (6 method precision and 6 intermediate precision) should not be more than 2.0%.

Validation Parameters

Results

Results Accuracy Level 50 % 100 % 150 % Overall % Recovery

Average % Recovery 99.2 100.2 100.5 100.0 %

%RSD 0.3 0.2 0.3

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Quantification of Busulfan in Pharmaceutical Formulations by Analytical HPLC (GRDJE/ Volume 6 / Issue 3 / 004)

–

The RSD at each level and overall RSD of % recovery should not be more than 5.0%

Overall % RSD

Condition

Robustness

System suitability criteria defined in test procedure should meet in each condition.

– The Tailing factor for Busulfan should be NMT 2.0.

– The relative standard deviation for Busulfan peak from five replicate injections of standard solution should be NMT 2.0 %. The theoretical plates for Busulfan peak in standard solution should be not less than 2000.

As such (For Flow, Temperature, Organic composition,Derivatisation temperature,Derivatisation Time) Flow rate:1.3 mL/min Flow rate:1.7 mL/min Column oven temperature: 23°C Column oven temperature: 27°C Low organic composition(637 mL) High organic composition(663 mL) Derivatisation temperature: 50° C Derivatisation temperature: 70° C Derivatisation time: 10 min Derivatisation time: 30 min

0.6 %

% RSD

Busulfan Tailing Theoretical plates factor

0.3

1.0

16290

0.2 0.3

1.0 1.0

20283 19156

0.3

1.0

20075

0.1 0.1 0.1 0.7 0.2 0.4 0.1

1.0 1.0 1.0 1.0 1.0 1.0 1.0

20145 19366 20976 19952 19793 20008 19837

X. FINAL CONCLUSION A simple isocratic HPLC method is developed for the determination of Busulfan in pharmaceutical formulations. The result meets the acceptance criteria and found comparable, indicates that the method is precise and rugged with respect to analyst to analyst, day to day, column to column and equipment to equipment for its intended use. Therefore the method can be used for routine analysis in quality control.

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