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

K. Balaji K. Raghu Babu

Assistant Professor Professor
Department of Chemistry Department of Chemistry
MVR College, Visakhapatnam, Andhra Pradesh 530045 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, 4-
methanesulphonyloxybutanol, 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

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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. Equipment Model Make/Supplier
1 Weighing Balance XS205 Dual Range Mettler Toledo
2 High Performance Liquid Chromatography LC-2030C Shimadzu
3 Ultrasonic cleaner 101/250 PCI Analytics
4 Micropipette SL-1000 Rainin
5 pH Meter PICO + Labindia

IV. CHEMICALS / CONSUMABLES

S.No. Name Grade Manufacturer
1 Acetonitrile High Performance Liquid Chromatography Merck Limited
2 Sodium diethyl dithiocarbomatetrihydrate High Performance Liquid Chromatography Merck Limited
3 N,N Dimethyl acetamide High Performance Liquid Chromatography Merck Limited
4 Tetrahydron Analytical Regent Grade 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
% Assay = -------- 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

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.
Table 1: Results of Linearity
Linearity Level Concentration (µg/mL) Area (Average)
50 % 27.1 1873386
75 % 36.9 2520268
100 % 49.2 3444590
125 % 61.5 4186867
150 % 73.8 4948738
Correlation coefficient (R) : 0.999
Slope : 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.
Table 2: Stability of Standard Solution
%Assay of Busulfan % Difference
Time Interval
Room Temperature 2-8°C Room Temperature 2-8°C
Initial 100 NA
24 hours 100.3 100.3 -0.30 -0.30
48 hours 101.3 101.3 -1.30 -1.30
Table 3: Stability of Sample Solution
%Assay of Busulfan % Difference
Time Interval
Room Temperature 2-8°C Room Temperature 2-8°C
Initial 100.5 NA
24 hours 99.7 101.3 0.80 -0.80
48 hours 101.0 102.5 -0.50 -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
Acceptance Criteria Results
Parameters
Error! Reference source not found. Component name % RSD
The relative standard deviation for Busulfan peak
area from five replicate injection of standard Busulfan 0.3%
solution should be not more than 2.0%
Precision
Error! Reference source not found.
The relative standard deviation of assay results
Busulfan 0.1%
obtained from six sample preparations should not
be more than 2.0%
Interference
There is no interference is observed at the
No Interference should be observed at the
retention time of Busulfan peak in the
Specificity retention time of Busulfan peak in the
chromatogram obtained from the diluent,
chromatograms obtained from the diluent, Blank
Blank and placebo.
and placebo.
Validation
Acceptance Criteria Results
Parameters
Drug Product (FP)
Error! Reference source not found. % Purity Purity
Array of Stress
Calculate the % degradation against degradation Angle Threshold
as such test preparation for each As Such (Unstressed) - 0.681 19.281
condition, in any of one condition Acid degradation 2.2 0.793 17.890
degradation should be achieved Alkali degradation 8.6 0.796 18.528
between 5.0% to 20.0%. Oxidation degradation 2.2 1.069 66.090
Each degradation sample, purity UV degradation Not degraded 0.690 18.648
angle should be less than the purity Thermal degradation 2.6 0.678 19.149
threshold for Busulfan peak. Neutral degradation 1.7 0.747 18.585
– Correlation coefficient should Busulfan
not be less than 0.999 for Correlation coefficient (R) 0.999
Busulfan. slope of regression line 66205
– Report the slope of regression Y-intercept of regression line 104381
line.
Linearity
– Report the Y-intercept of
regression line.
– Y-intercept bias at 100 % level Y-intercept bias at 100% level 3.0
should be between ± 5.0 % for
Busulfan.
– The relative standard deviation Precision Intermediate Precision
results obtained from six 0.1% 0.6%
sample preparations should not
be more than 2.0%
– The cumulative %RSD of
Intermediate method precision and
Precision intermediate precision results
0.4%
obtained from twelve sample
preparations (6 method
precision and 6 intermediate
precision) should not be more
than 2.0%.
Validation
Acceptance Criteria Results
Parameters
Accuracy Level Average % Recovery %RSD
– Recovery at each level and
50 % 99.2 0.3
overall average recovery of
100 % 100.2 0.2
assay results should be between
150 % 100.5 0.3
98.0% and 102.0%
Accuracy Overall % Recovery 100.0 %

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 Quantification of Busulfan in Pharmaceutical Formulations by Analytical HPLC
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– The RSD at each level and

overall RSD of % recovery
Overall % RSD 0.6 %
should not be more than 5.0%

Busulfan
Condition % Tailing
Theoretical plates
System suitability criteria defined in RSD factor
test procedure should meet in each As such (For Flow, Temperature,
condition. Organic composition,Derivatisation 0.3 1.0 16290
– The Tailing factor for Busulfan temperature,Derivatisation Time)
should be NMT 2.0. Flow rate:1.3 mL/min 0.2 1.0 20283
– The relative standard Flow rate:1.7 mL/min 0.3 1.0 19156
Robustness deviation for Busulfan peak Column oven
0.3 1.0 20075
from five replicate injections temperature: 23°C
of standard solution should be Column oven temperature: 27°C 0.1 1.0 20145
NMT 2.0 %. The theoretical Low organic composition(637 mL) 0.1 1.0 19366
plates for Busulfan peak in High organic composition(663 mL) 0.1 1.0 20976
standard solution should be Derivatisation temperature: 50° C 0.7 1.0 19952
not less than 2000. Derivatisation temperature: 70° C 0.2 1.0 19793
Derivatisation time: 10 min 0.4 1.0 20008
Derivatisation time: 30 min 0.1 1.0 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.

REFERENCES
[1] P.W. Feit, N. Rastrup-Andersen, J. Pharm. Sci. 62: p1007, 1973.
[2] US Food and Drug Administration Busulfex New Drug Application (NDA): 020954, 1999.
[3] J.Pidala, J.Kim, C. Anasetti, Kharfan-Dabaja, M.A. T. Nishihori, et al. “Pharmacokinetic targeting of intravenous busulfan reduces conditioning regimen
related toxicity following allogeneic hematopoietic cell transplantation for acute myelogenous leukemia”. Journal of Hematology & Oncology 3: p36, 2010.
[4] I.H. Bartelink, R.G. Bredius, T.T. Ververs, M.F. Raphael, C. van Kesteren,et al. “Once-daily intravenous busulfan with therapeutic drug monitoring compared
to conventional oral busulfan improves survival and engraftment in children undergoing allogeneic stem cell transplantation”. Biology of Blood and Marrow
Transplantation 14: p88-98, 2008.
[5] J. Zwaveling, J. Den Hartigh, A.C. Lankester, H.J. Guchelaar, R.M. Egeler, et al. “Once-daily intravenous busulfan in children prior to stem cell
transplantation: study of pharmacokinetics and early clinical outcomes”. Anticancer Drugs 17: p1099-1105, 2006.
[6] M. Lesurtel, R. Graf, B. Aleil, D. Walther, Y. Tian, W. Jochum, C. Gachet, M. Bader, P. Clavien. "Platelet-derived serotonin mediates liver regeneration".
Science. 312 (5770): p104–7, 2006.
[7] A. Grigg, R. Gibson, P. Bardy, J. Szer. "Acute portal vein thrombosis after autologous stem cell transplantation". Bone Marrow Transplant. 18:p949–53,
(1996).
[8] H. Brisse, D. Orbach, N. Lassau, V. Servois, F. Doz, D. Debray, S. Helfre, O. Hartmann, S. Neuenschwander. "Portal vein thrombosis during antineoplastic
chemotherapy in children: report of five cases and review of the literature". Eur. J. Cancer. 40: p2659–66, 2004.
[9] H.P. Bhagwatwar, S. Phadungpojna, DS-L. Chow, B.S. Andersson. "Cancer Chemother Pharmacol," 37: p401-8, 1996.
[10] B.S. Andersson, T. Madden, H.T. Tran, et al. "Biol Blood Marrow Transplant", 6: p548-54, 2000.
[11] B.S. Andersson, A. Kashyap, V. Gian, et al. "Biol Blood Marrow Transplant",8: p145-54, 2002.
[12] Andrew Teasdale, David Elder, Raymond W Nims, ICH Quality Guidelines: An Implementation Guide, ISBN: 978-1-118-97111-6, 2017.
[13] Khagga Bhavyasri , Kaitha Manisha Vishnumurthy, Dammu Rambabu, and Mogili Sumakanth, ICH guidelines – “Q” series (quality guidelines), GSC
Biological and Pharmaceutical Sciences, 06, p089–106, 2019.

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