INTRODUCTION:

Clofarabine (CAS No. 123318-82-1) is an anti-cancer drug¹. In 2004, the Food and Drug Administration (FDA) has approved. Clofarabine (Figure 1) is a purine nucleoside used to treat relapsed or refractory acute lymphoblastic leukemia in patients 1 to 21 years old. Chemically Clofarabine (CLO) is 5-(6-amino-2-chloro-purin-9-yl) -4-fluoro-2- (hydroxymethyl) oxolan-3-ol with molecular formula, C₁₀H₁₁ClFN₅O₃ and molecular weight 303.68 grams/mole.

Figure 1: Structure of Clofarabine (C₁₀H₁₁ClFN₅O₃)

Clofarabine was earlier studied by different analytical techniques such as HPLC^2-4, UPLC⁵, LC-MS/MS⁶ in formulations as well as biological fluids such as plasma, urine etc.

In the present study a new stability indicating HPLC and LC-APCI-MS methods have been proposed for the quantification of Clofarabine and the method was validated as per ICH guidelines.

MATERIALS AND METHODS:

Instrumentation

HPLC Conditions

TSQ scientific Quantis LCMS with Thermo Vanquish model HPLC with PDA detector and Simpack C18 (250 mm x 4.6 mm x 5µm) column was employed for the present study. The injection volume was 10µL and the total run time was 25 mins (Detection wavelength 250 nm). 0.1% Formic acid: Acetonitrile was used as mobile phase on gradient mode with flow rate was 1 mL/min and a mixture of Methanol: Water (50:50) was used as diluent.

MS Conditions

Ion Source type : APCI

Spray Voltage : Static

Positive Ion discharge current (V) : 4

Negative Ion discharge current (V) : 10

Sheath Gas (Arb) : 45

Aux Gas (Arb) : 10

Sweep Gas (Arb) : 2

Ion transfer tube temperature : 275

Vaporizer temperature : 400

Scan mode : Full scan Q1

Scan Range : 50-2000 m/z (Positive mode)

: 50-2000 m/z (Negative mode)

Preparation of stock solution

25mg of Clofarabine API was weighed accurately and transferred carefully into a 25mL volumetric flask and was dissolved in HPLC grade Acetonitrile (1000 µg/mL) and the resulting solution was sonicated for 30 mins.

Method validation⁷

Linearity, Precision, Accuracy and Robustness

2.0-200 µg/mL Clofarabine solutions were prepared from the stock solution (1000 µg/mL) on dilution with the mobile phase and each solution was injected (n=3) into the LC system and the average peak area from the respective chromatograms was calculated. A calibration graph was drawn by plotting the concentration of the drug solutions on the x-axis and the corresponding peak area of the chromatograms on the y-axis. The intraday precision studies were conducted on the same day at different equal time intervals and the interday precision studies were conducted on three successive days (Day 1, Day 2 and Day 3) and the % RSD was calculated.

Accuracy studies were performed by spiking the formulation solution with 50, 100 and 150% API solution and thereby the percentage recovery was calculated with the help of regression equation. The percentage relative standard deviation was calculated in all the validation parameters.

Assay of Clofarabine

Clofarabine is available as intravenous injection with label claim: 20 mg/20 mL with brand names such as FARABINE (Intas Pharmaceuticals), CLOLAR (Generic) and Cfara (Label (Zydus) in India. Two different brands of Clofarabine were collected and extracted with acetonitrile and after sonication diluted with the diluent as per the requirement. The resulting solution was filtered through 0.24μm membrane filter and 10μL of these formulation solutions were injected in to the HPLC system. The peak area of the chromatogram (n =3) was noted and the percentage purity was determined.

Stress degradation studies⁸

During the acidic degradation study Clofarabine solution was treated with 0.1N HCl and immediately neutralized with 1mL 0.1N NaOH solution. The contents were diluted with mobile phase and the resultant solution was injected into HPLC and LC-MS system and the peak area as well as the mass spectrum was recorded.

During the thermal degradation study Clofarabine solution was heated at 60ºC and the contents were diluted with mobile phase and the resultant solution was injected into HPLC and LC-MS system and the peak area as well as the mass spectrum was recorded.

During the basic degradation study Clofarabine solution was treated with 0.1N NaOH for about 30 mins and then neutralized with 1mL 0.1N HCl solution. The contents were diluted with mobile phase and the resultant solution was injected into HPLC and LC-MS system and the peak area as well as the mass spectrum was recorded.

During the oxidative degradation study Clofarabine solution was treated with hydrogen peroxide for about 30 mins and then diluted with mobile phase and the resultant solution was injected into HPLC and LC-MS system and the peak area as well as the mass spectrum was recorded.

RESULTS AND DISCUSSION:

A new stability-indicating RP-HPLC and LC-MS methods have been developed for the quantification of Clofarabine. The earlier reported methods were discussed with the present proposed method and the details were given in Table 1.

Table 1: Literature survey

Method	Mobile phase (v/v)	Linearity (μg/mL)	Reference
RP-HPLC (Isocratic mode)	Tri fluoro acetic acid buffer (pH 3.6): Methanol: Acetonitrile (70:15:15)	10-30	2
RP-HPLC (Isocratic mode)	Buffer: Acetonitrile (90:10)	5-25	3
RP-HPLC (Related impurities)	Phosphate buffer (pH 3.0): Acetonitrile (Gradient mode)	0.05-20	4
RP-UPLC (Related substances)	Ammonium formate buffer (pH 3.0): Acetonitrile (Gradient mode)	0.2-1.6	5
LC-MS/MS (Urine and Plasma)	Methanol: 1 mM ammonium acetate (Gradient mode)	0.002-1.0	6
RP-HPLC and LC-APCI-MS	0.1% Formic acid: Acetonitrile (Gradient mode)	5.0-150	Present method

TSQ scientific Quantis LCMS with Thermo Vanquish model HPLC with Simpack C18 (250 mm x 4.6 mm x 5µm) column, PDA detector, APCI and triple quadrupole analyser was employed for the present study. The injection volume was 10 µL and the total run time was 25mins (Detection wavelength 250 nm). Mobile phase consisting of 0.1% Formic acid: Acetonitrile (A: B) was used on gradient mode (Table 2) with flow rate was 1mL/min and a mixture of Methanol: water (50:50) was used as diluent.

Table 2: Gradient program

Time (min)	Mobile phase A%	Mobile phase B%
0.0	95	5
5.0	95	5
15.0	10	90
20.0	10	90
20.1	95	5
25.0	95	5

Clofarabine was eluted at Rt 11.967 min with theoretical plates more than 2000 and tailing factor less than 1.5. The HPLC and LC-MS chromatograms and mass spectra of Clofarabine obtained in the optimized chromatographic conditions were shown in Figure 2.

Linearity, Precision, accuracy and robustness

Clofarabine obeys Beer-Lambert’s law over the concentration range 5.0-150 µg/mL (Table 3) and the linear regression equation was found to be y = 188.07x + 125.28 (R² = 0.9996) Figure 3). The LOD and LOQ values were found to be 1.5874µg/mL and 4.8213µg/mL respectively. The % RSD in intraday precision (0.0827), interday precision (0.0168-0.9191) (Table 4) was found to be less than 2.0% stating that the method is precise. In the accuracy study the % RSD was found to be 0.29-0.92 (<2) (Table 5) with a recovery of 99.63-99.81 indicating that the method is accurate.

Table 3: Linearity

Conc. (µg/mL)	*Mean peak area
0	0
5	1022.128
10	1929.834
25	4825.913
50	9539.251
75	14464.986
100	19275.842
150	27991.568

*Mean of three replicates

Figure 3: Calibration curve

Table 4: Precision study

Intraday precision study
Conc. (µg/mL)	Mean peak area		*Mean peak area ± SD (% RSD)
10	1929.834		1928.3062 ± 1.5953 (0.0827)
10	1928.247
10	1928.692
10	1929.192
10	1925.248
10	1928.624
Interday precision study
Conc. (µg/mL)	Day 1	Day 2	Day 3	Mean peak area ± SD (% RSD)*
10	1929.834	1927.121	1931.258	1929.4043 ± 2.1017 (0.1089)
50	9539.251	9601.101	9713.615	9617.989 ± 88.4003 (0.9191)
100	19275.842	19282.257	19279.918	19279.339 ± 3.2465 (0.0168)

*Mean of three replicates

Table 5: Accuracy study

Spiked conc. (µg/mL)	Formulation (µg/mL)	% Recovery	% RSD
25 (50 %)	50	99.76	0.29
50 (100 %)	50	99.63	0.71
125 (150 %)	50	99.81	0.92

*Mean of three replicates

Assay of Clofarabine

The assay of Clofarabine intravenous injection was performed using the proposed liquid chromatographic method with the optimized chromatographic conditions. The percentage of purity of Clofarabine was found to be 99.59-99.83 (Table 6).

Table 6: Assay of Clofarabine

S. No.

Brand name

Label claim

(mg/mL)

*Observed amount

(mg/mL)

% Recovery*

Brand I

0.9959

99.59

Brand II

0.9983

99.83

*Mean of three replicates

Stress degradation studies

Clofarabine (100 µg/mL) was exposed to different stress conditions under the optimized chromatographic conditions and then injected in to the system. During the acidic degradation, Clofarabine was eluted at Rt 11.808 min and about 14.16 % has undergone decomposition. The mass spectra at Rt 11.91 min and 11.94 min were shown in Figure 4.


Acid blank	Representative chromatogram of Clofarabine (Rt 11.808 min) during acidic degradation

LC-MS Chromatograms of Clofarabine during acidic degradation

Mass spectrum of Clofarabine during acidic degradation (Rt 11.91 min; MH⁺: m/z 304.07)	Mass spectrum of Clofarabine during acidic degradation (Rt 11.94 min)

Figure 4: Representative chromatograms and mass spectra of Clofarabine during acidic degradation

During the thermal degradation, Clofarabine was eluted at Rt 11.817 min and about 14.74 % has undergone decomposition. The mass spectra at Rt 11.91 min and 11.88 min were shown in Figure 5.


Representative chromatogram of Clofarabine (Rt 11.817 min) during thermal degradation

LC-MS Chromatograms of Clofarabine during thermal degradation


Mass spectrum of Clofarabine during thermal degradation (Rt 11.91 min)	Mass spectrum of Clofarabine during thermal degradation (Rt 11.88 min)

Figure 5: Representative chromatograms and mass spectra of Clofarabine during thermal degradation

During the basic degradation, Clofarabine was eluted at Rt 11.800 min and about 4.48 % has undergone decomposition. The mass spectra at Rt 11.94 min and 11.98 min were shown in Figure 6.


Base blank	Representative chromatogram of Clofarabine (250 nm) during basic degradation

LC-MS Chromatogram of Clofarabine during basic degradation

Mass spectrum of Clofarabine (Rt 11.98 min) during basic degradation	Mass spectrum of Clofarabine (Rt 11.94 min) during basic degradation

Figure 6: Representative chromatograms and mass spectra of Clofarabine during basic degradation

During the oxidative degradation, Clofarabine was eluted at Rt 11.800 min and about 4.91 % has undergone decomposition. The mass spectra at Rt 10.100, 11.008, 11.092, 11.350, 11.633 min were shown in Figure 7.


Peroxide blank	Representative chromatogram of Clofarabine (Rt 11.800 min) during peroxide degradation

LC-MS chromatogram of Clofarabine (250 nm) during peroxide degradation

Mass spectrum of Clofarabine (Rt 11.91 min) during oxidative degradation	Mass spectrum of Clofarabine (Rt 11.94 min) during oxidative degradation

Figure 7: Representative chromatograms and mass spectra of Clofarabine during oxidative degradation

Table 7: Stress degradation studies

Condition	R_t (min)	*Mean peak area	% Recovery*	% Drug degradation	Resolution
Standard drug	11.817	19275.842	100	-	-
Acidic hydrolysis	11.808 11.333	16487.680	85.54	14.46	3.62
Thermal degradation	11.817 11.342	16435.282	85.26	14.74	4.03
Alkaline hydrolysis	10.042 11.800 11.083	18412.799	95.52	4.48	5.86
Oxidative degradation	10.100 11.008 11.092 11.350 11.633 11.800	18329.394	95.09	4.91	2.88 2.34 1.30

*Mean of three replicates

The details of the stress degradation studies of Clofarabine were shown in Table 7. It is observed that Clofarabine is highly resistant towards all degradation conditions.

CONCLUSION:

The authors have established a new stability indicating RP-HPLC as well as LC-MS method coupled with APCI and triple quadrupole analyser for the estimation of Clofarabine. The method is simple, precise and accurate and used for the routine analysis of Clofarabine in pharmaceutical formulations and no interference of excipients was observed during the assay.

ACKNOWLEDGEMENT:

The authors are grateful to MSN Laboratories Pvt. Ltd. (India) for providing the gift samples of Clofarabine and the authors declare no conflict of interest.

REFERENCES:

1. Nijstad AL, Nierkens S, Lindemans CA, Boelens JJ, Bierings M, Versluys AB, van der Elst KCM, Huitema ADR. Population pharmacokinetics of Clofarabine for allogeneic hematopoietic cell transplantation in paediatric patients. Br J Clin Pharmacol. 2021; 87(8): 3218-3226.

2. Chaithanya Sudha PD. Validated RP-HPLC method for the determination of Clofarabine in bulk and tablet dosage. /Journal of Pharmaceutical Sciences and Research. 2019; 11(5): 1781-1786.

3. Jinal N Tandel, Jimi N Patel and Samir K Shah. Quantification of Clofarabine and its impurity substances by RP-HPLC method in parenteral formulation. International Journal of Pharmaceutical Sciences and Nanotechnology. 2017; 10(4).

4. Jagadeswara Rao K, Murali Mohan SV and Rama Rao Malla. Validated Reverse Phase Stability-Indicating HPLC Method for Clofarabine in the Presence of Degradation Products and its Process-Related Impurities. 2016; 11(6): 63-72.

5. Dhananjay Prasad D, Ravi Kumar B, Sidda Reddy K and SreeramLu J. Ultra-performance liquid chromatographic method for quantification of Clofarabine related substances in an injection formulation. International Journal of Innovative Science and Research Technology. 2017; 2(7): 334-345.

6. Zhang X, Jia X, Tong W, Chen H, Lei N, Li G, Tai J, Li P. Quantification of clofarabine in urine and plasma by LC-MS/MS: suitable for PK study and TDM in pediatric patients with relapsed or refractory ALL. RSC Adv. 2022; 12(51): 33091-33098.

7. ICH Validation of analytical procedures: Text and methodology Q2 (R1), International Conference on Harmonization (2005).

8. ICH Stability testing of new drug substances and products Q1A (R2), International Conference on Harmonization (2003).

Received on 24.03.2023 Modified on 29.04.2023

Research J. Pharm. and Tech 2023; 16(5):2485-2491.

DOI: 10.52711/0974-360X.2023.00409


Blank	Representative HPLC chromatogram of Clofarabine (Rt 11.817 min)

LC-MS Chromatograms of Clofarabine (API)	Mass spectrum of Clofarabine Rt 11.91 min

Mass spectrum of Clofarabine Rt 11.94 min

GITAM School of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India.