Residual Solvents in Bendamustine Hydrochloride By Headspace Chromatography

 

J. Ramesh Babu, J. Suhasini, S. Vidyadhara

Chebrolu Hanumaiah Institute of  Pharmaceutical Sciences, Chandramoulipuram, Chowdavaram, Guntur.(A.P.), India.

*Corresponding Author E-mail: ramesh.janga11@gmail.com

 

ABSTRACT:

There are only few published reports on determination of residual solvents in the analytical method development and there exists no detailed guidelines. Residual solvents from the process in the manufacturing of pharmaceuticals are hazardous and cause serious problems, so must be removed. This is much effort in this work is focused on the determination of analytical method for the determination of residual solvents. Bendamustine hydrochloride pure drug, various solvents, Shimadzu GC-2010 with head space auto injector and AOC – 5000 with head space auto injector were used. The method development based on residual solvent properties and many trails conducted on conditions like column selection, carrier gas flow, oven temperature and diluent. A simple HS-GC method for the determination of residual solvents in Bendamustine hydrochloride using helium as the carrier gas at 3.0mL/min with DB-624 (30.0 meters X 0.53 mm ID, 3.0µm d.f. capillary) as column using FID as detector was developed. The developed method was validated and parameters were to be found within the limits of USP. The retention time for residual solvents individually and in spiked standard solution was determined. The %RSD for six injections should be NMT15%. The percentage recovery ranges from 84.57-118.86%. The correlation coefficient was R2 ≥ 0.999. The method was validated for repeatability, linearity, limit of detection, limit of quantification and recovery according to the International Conference on Harmonization guidelines. The method validation results indicate that the method is accurate, precise, linear and sensitive for solvents assessed. Excellent results were obtained, within the globally accepted validation reference values, particularly taking into account the low concentration levels investigated.

 

KEYWORDS: Validation, Residual solvents, HS-GC and Bendamustine hydrochloride.

 

 


 

INTRODUCTION:

Residual solvents, or organic volatile impurities, are a potential toxic risk of pharmaceutical products and have been a concern of manufacturers for many years [1]. Moreover, residual solvents can also affect the quality and stability of not only drug substances but also drug products [2,3]. Thus, acceptable levels of many are included in regulatory guidance documents; in particular in guideline Q3C issued by the International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human use (ICH)[4]. Residual solvents are classified into four classes on the basis of the toxicity level and the degree to which they can be considered an environmental hazard [5]. Class 1 solvents are known carcinogens and are strongly suspected of being harmful to humans and the environment, so they should be avoided. Class 2 solvents are nongenotoxic animal carcinogens. Solvents of this class should be limited in pharmaceutical products because of their inherent toxicity. Class 3 solvents have low toxic potential to humans and should be used only where it would be impractical to remove them. Finally, Class 4 solvents are those for which no adequate toxicological data have been found. These last three classes of solvents are the ones most commonly analyzed. Residual solvents are typically determined using chromatographic techniques such as static headspace gas chromatography (HSGC)[ 6]. Developing and validating an efficient and sensitive generic analytical method for the determination of residual solvents may significantly increase productivity of an analytical laboratory in the pharmaceutical Industry. Determination of residual solvents using GC with a flame ionization detector (FID) is the most common technique in the pharmaceutical industry, because of its high separation efficiency and sensitivity for volatile organic compounds. Headspace gas chromatography (HS-GC) method has been used for the determination of residual solvents in pharmaceutical compounds [7,8]. Direct injection of analytes evaporated through equilibration between liquid (or solid) phase and gas phase to GC system minimized the contamination of GC system and the deterioration of GC column [9,10] . Volatile residual solvents are accumulated prior to analysis.

 

Here I report a full validation of a HS-GC analytical method for determination of five residual solvents (Class Bendamustine hydrochloride, methnol, ethanol, acetone, isopropyl alcohol and toluene) commonly used during the manufactories of drug substances and purification steps and its dosage forms.

 

MATERIALS AND METHODS:

Instrumentation and Chromatographic conditions:

The analysis was performed on Shimadzu Gas Chromatography; Japan equipped with model no Shimadzu - GC - 2010 head-space AOC 5000 auto sampler and a flame-ionization detector. The injector temperature was 100CC and detector temperature was 250oC. Column was DB-624 with serial no-US7109941H (100% dimethylpolysiloxane 30.0 m × 0.53 mm ID, 3.0 μm d.f. Capillary). Split ratio of injection 1:2. Initially kept at 40°C for 6 min, raised to 130°C @ 10°C/min hold 8 min. Raised to 240°C @ 35°C/min hold 5 min. Total run time was 22 min. Helium was used as a carrier gas at a constant flow rate of 3.0 ml/min.

 

Table No 1.

S.No

Name

Grade

Supplier

LOT No(or) B.No

Assay (or) Purity

1

Methanol

AR

Rankem

R153A12

99.0%

2

Ethanol

AR

Heyman

13/528A6

99.9%

3

Isopropyl alcohol

AR

Rankem

R026K12

99.9%

4

Chloroform

AR

Rankem

B112A13

99.4%

5

Toluene

AR

Rankem

B014MO12

99.5%

6

Dimethyl formamide

AR

Rankem

R130F13

99.5%

7

Bendamustine. HCl

IH

Natco

BND/L/015/13

99.5%

 

Method: Head space Gas chromatography

The analysis was performed on Shimadzu GC-2010 with head space auto injector and FID detector with helium as the carrier gas. The chromatographic conditions are given in Table No 2.

 

Table No 2:

Head space conditions

Column                                       

OVI-G43 (30mm x 0.53mm and 3.0µm)

Oven temperature

800C

Loop temperature

900C

Transfer line temperature             

100oC

Equilibration time                          

20 minutes

Pressurization time                     

0.3 minutes

Loop full time

0.2 minutes

Loop equilibration time                  

0.05 minutes

Injection time Injection volume     

0.5 minutes

Injection Volume                          

1.0ml (loop) auto injection system

GC cycle                                        

25 minutes

Instrument Parameters:

Oven temperature                       

T1: 450C, t :10 minutes, Rate : 15oC/min

T2: 750C, t :2 minutes, Rate : 25oC/min

 T3: 2000C, t3:3 minutes       

Injector temperature                    

200oC

Detector temperature                  

250oC

Carrier gas (Helium)                   

3.0ml/min

Split ratio                                       

1:2

Run time                                      

22 minutes

 

Preparation of standard solution:

100ml volumetric flask, half filled with diluents (DMF) was taken  and  accurately  weighed amount of  750mg of Methanol, 1250mg of Ethanol, 1250mg of Isopropyl Alcohol, 15mg of Chloroform and 222.5mg of Toluene, then diluted to volume with diluent. 5ml of stock solution was transferred into 50ml volumetric flask, half filled with diluent and dilute the volume with diluent.

 

Sample preparation Weigh approximately 250mg of sample and transfer to a 20mL headspace vial add 2 ml of diluents.

Procedure:

Transfer the above prepared standard solutions each 2 ml into six different HSS vials and sealed with aluminum closure. Each of the vials contains 500 ppm of methanol, 500 ppm of ethanol, 500 ppm of isopropyl alcohol, 500 ppm of chloroform and 100 ppm of toluene with respect to the sample. The vials have a DMF solution containing solvents at different concentrations, the vials are kept at 45°C the headspace sampler was equipped with a 1-mL sample loop. Since a sufficient flow must be maintained through the system to avoid excessive peak broadening.

 

 

Figure 1: chromatogram of Bendamustine hydrochloride

 

 

Figure 2: chromatogram for system suitability ( Bendamustine hydrochloride)

 

 

Method Validation:

The parameters like specificity, accuracy, LOD and LOQ, system suitability were performed that are mentioned in the International conference on harmonization (ICH) guidelines. Specificity is performed to know the retention time further residual solvents individually and in spiked sample solution. Linearity was done to know the test results which are directly proportional to the concentration of analyte in the sample. It was performed from LOQ to 150% and results were found to be within the limits. Precision was validated to know the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample. %RSD for precision was also found to be NMT 15%.

 

Accuracy is the amount of drug recovered from the spiked sample. It is assessed by 9 Determinations over a minimum of 3 concentration levels covering the specified range. Robustness is tested by introducing small variations in method parameters. From the results it was observed that the method remain unaffected. System suitability is performed to ensure that the complete testing system is suitable for Intended application finally the sample is checked for the presence of residual solvents in Bendamustine hydrochloride.

 

RESULTS:

In this study, a HS-GC analytical method was developed and validated for the quantitative determination of the solvents methanol, ethanol, isopropyl alcohol, chloroform and toluene in Bendamustine hydrochloride. The proposed method uses the standard addition technique with internal standard quantitation for determination of residual solvents. The method was validated within ICH guidelines Q2A and Q2B. Selectivity, limits of detection and quantitation, linearity, range, precision (system repeatability), recovery and robustness (changes in HS and GC conditions and solution stability) were determined. Excellent results were obtained, within global validation reference values, particularly taking into account the low concentration levels investigated. The test method was validated and had good reproducibility and linearity for the solvents used in the manufacturing process. The recovery was good and justified the preparation of the standard in DMF without the product as matrix.

 

The concentration of residual solvents (ppm) in the drug samples can be determined using the formula:


=

Area of individual solvents in test solutions

×

Individual solvent weight in standard solution (mg)

×

10

×

10

×

106

Average area (six injections) of individual solvent in standard solution

Wt.of sample in (mg)

100

100

 


The linear range and correlation coefficients were determined between 10 ppm and 1800 ppm. The results for the 2-ml sample volume are documented in table-4.

 

Selectivity:

The ZB-624 column, in the 30 m x 0.32 mm I.D. configuration, was chosen because this column has a standard stationary phase, which is recommended by the European and American Pharmacopeias, and has provided baseline separations of all solvents used in the validation, including the diluent (DMF). The method showed good peak shape, and the narrow peak width resulted in excellent column efficiency. The blank chromatogram did not show any interference with the solvent peaks.


 

Table No 3:


 

Linearity

Accuracy

Solvents

Range (%)

R2

Slope

Recovery (%)

Average value (ppm)

Average

RSD (%)

Methanol

 

 

10-150

0.9996

2.1565

83.10-91.95

2602.1

83.1

3.60

Ethanol

0.9993

1.9482

85.46-102.12

4759.5

 

5.49

Isopropyl alocohol

0.9994

2.0809

99.86-117.69

5212.4

99.86

3.63

Chloroform

0.9997

0.5457

95.51-111.86

91.6

95.51

2.94

Toulene

0.9996

4.3383

104.85-118.26

1578.8

104.85

3.23

 


Table No 4: summary of validation parameters for the proposed method

Specificity

No interference was found W.R.T ecipients

Linearity (R)

0.999

Range

LOQ – 150%

Precision (%RSD)

a. system suitability

b. system precision

c. method precision

 

1.41-1.92

2.87-4.51

0.18-2.05

Accuracy (% Recovery)

84.57-118.86%

LOQ

2.63-9.36

Ruggedness

0.54-1.52

Robustness

1.63-2.15

Solution stability

No interference was found W.R.T Recipients

 

Linearity and range:

To carry out this study, six concentrations were prepared of each solvent. All concentrations were prepared in triplicate, by individually weighing amounts of solvents. The experimental results were represented graphically to obtain a calibration curve and carry out the corresponding statistical study (Anova). The method is linear within a wide range for the solvents included in the validation. The correlation coefficients were all above 0.999 and linear regression showed a positive response throughout the range (Fig 3-7).

 

The specified range is normally derived from linearity studies and depends on the intended application of the procedure. The wide measurement range allows determination with adequate precision of different analyte contents in various matrices. The measurement ranges are shown in the Table 3 and 4 with the respective RSD values.

 

Repeatability:

Repeatability was determined in accordance with ICH guidelines, i.e.: nine independent determinations were carried out during single day and on their basis the values of the standard deviations were established. The repeatability, representing the spread of the results, was expressed as RSD (Table 4).

 

 

Figure 3: Calibration curve for Methanol

 

 

Figure 4: Calibration curve for Ethanol

 

 

Figure 5: Calibration curve for Isopropyl alcohol

 

 

Figure 6: Calibration curve for Chloroform

 

 

Figure 7: Calibration curve for Toulene

 

Figure 8: Chemical structure of Bendamustine hydrochloride

 

 

CONCLUSION:

The analytical method proposed for the quality control Bendamustine hydrochloride in relation to the residual methanol, ethanol, isopropyl alcohol, chloroform and toluene contents, met the validation requirements. Excellent results were obtained, within globally accepted validation reference values, particularly taking into account the low concentration levels investigated. The method was sensitive, linear, accurate and precise. Three randomly selected batches of each drug substance were analyzed under validated method conditions and the concentrations of residual methanol, ethanol, isopropyl alcohol, chloroform and toluene were much lower than their maximum ICH limits. Moreover, the validated method can be applied to others drug substances.

 

REFERENCES:

1.     Sitaramaraju,Y; Riadi, A.; D’Autry,W; Wolfs, K.; Hoogmartens, J.; Schepdael, A. V.; Adams, E. Static headspace gas chromatography of (semi-) volatile drugs in pharmaceuticals for topical use. J. Pharm. Biomed. Anal., 2008, 48, 113.

2.     Faria, A. F.; Souza, M. V. N.; Oliveira, M. A. L. Validation of a Capillary Zone Electrophoresis Method for the Determination of Ciprofloxacin, Gatifloxacin, Moxifloxacin and Ofloxacin in Pharmaceutical Formulations. J. Braz. Chem. Soc., 2008, 19,. 389,

3.     Antolín, E. M.; Quinónez,Y. B.; Canavaciolo,V.G.; Cruz, E. R. Validation of an analytical method for quality control of residual solvents (n-hexane and acetone) in D-002: new active ingredient from beeswax. J. Pharm. Biomed. Anal. 2008, 47, 646.

4.     Proceedings of International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), Tripartite harmonized guideline 3C “Impurities: Residual Solvents” 1997.

5.     Nojavan, S.; Ghassempour, A.; Bashour, Y.; Darbandi, M. K.; Ahmadi, S. H.; Determination of residual solvents and investigation of their effect on ampicillin trihydrate crystal struct. J. Pharm. Biomed. Anal., 2005, 36, 983.

6.     Gomes, P. C. F. L.; D’Andrea, E. D.; Mendes, C. B.; Siqueira, M. E. P. B. Determination of Benzene, Toluene and N-Hexane in Urine and Blood by Headspace Solid-Phase Microextration/Gas- Chromatography for the Biomonitoring of Occupational Exposure. J. Braz. Chem. Soc. 2010, 21,119.

7.     Mahesh, P.; Swapnalee, K.; Aruna, M.; Anilchandra, B.; Prashanti, S. Analytical Method Development And Validation Of Acetaminophen, Caffeine ,Phenylephrine HydrochlorideAnd Dextromethorphan Hydrobromide In Tablet Dosage Form By RP- HPLC. International J. of Pharmaceutical, 2013, 2, 2319 – 6718

8.     Liu, Y. and Hua, C.O., Establishment of a knowledge base for identification of residual solvents in pharmaceuticals. Anal Chem. Acta, 2006, 575.246.

9.     Clayton, B., Hymer. Residual solvent testing: A Review of Gas Chromatographic and Alternative techniques Pharm Res., 2003, 23, 337.

10.  Mary, A., Jack, H.U., Pengu, Z., Nina, C., Simultaneous determination of formic acid and formaldehyde in pharmaceutical excipients using head space GC/MS. J Chrom: Biomedical Appl ., 2006, 41, 783.

11.  ICH Guidelines Q2B, Analytical validation-methodology; 1996.

12.  ICH Topic Q3C (R4) Impurities: Guideline for Residual Solvents,European Medicines Agency CPMP/ICH/283/95, February 2009.

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

14.  International Conference on Harmonization, Impurities in new drug substances, 2002. Available from: http:// www.ich.org

 

 

 

 

 

 

 

 

 

Received on 28.08.2017          Accepted on 05.10.2017        

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(1):07-12.

DOI:    10.5958/2231-5675.2018.00002.9