INTRODUCTION:

Residual solvents in pharmaceuticals are defined as organic volatile chemicals that are used or produced in the manufacture of drug substances or excipients, or in the preparation of drug products¹. Determination of residual solvent is necessary because residual solvents can present potential risk to human due to their toxicity. It is also may cause changes in the physiochemical properties of the pharmaceutical product^2-7. Solvents are routinely used in the synthesis and process chemistry of drug substances. These solvents cannot be completely removed by practical manufacturing practices, and the residue should be within the normal limit⁸. European Pharmacopoeia classified the residual solvents in three classes, ethanol was classified in the third class of solvent, It is considered that an amount of this class of 50 mg per day or less (corresponding to 5,000 ppm or 0.5%, when the daily dose does not exceed 10 g) is permitted^2-9. Headspace gas chromatography (HS-GC) is an effective technique for measuring volatile chemicals in samples with complex matrices. The method is based on sampling the equilibrated vapor phase (headspace) above the liquid or solid sample in a closed vial, and then measuring the volatile species in the headspace by GC¹⁰.

It has been adopted, as a recommended method, by the pharmacopeias in the European Union (Ph.Eur.) and the United States (USP)^11-13. In the direct injection nonvolatile compounds or dissolution media would remain on the column, which could reduce the lifetime of the column and may cause contamination.

Thus, headspace analysis (HS) is thought to be more suitable technique for residual solvent testing which avoids many of the drawbacks of direct injection¹⁴. There are Two types of HS sampling techniques are generally available: static and dynamic. The static HS sampling technique is based on thermostatic partitioning of volatile compounds in a closed vial between the sample dissolution medium and the surrounding gas phase, followed by the transfer of an aliquot of the vial headspace gas containing the volatile analytes to the GC equipment¹⁵.

MATERIAL AND METHODS:

Reagents:

The following compounds were used during experiments:

Ethanol standard 99.9 from Dikma

Ethanol 96%

N,N-dimethylacetamide diluent

Methanol was used for cleaning laboratory glasses.

Distilled water was used to prepare sample and standard solutions.

Experimental:

Instrumentation:

Analysis of ethanol was carried out on gas chromatography from Agilent model 7890A equipped with flame ionization detector. Separations were performed on Dikma DM-5 capillary column (30m×0.25mm i.d) with a phase thickness of 0.25 µm film (5% dibenzyl-95% dimethylpolysiloxane). The extraction was performed using Headspace sampler with 20 ml vial.

The initial carrier gas flow was approximately 0.4 ml/min.

Chromatographic conditions are presented in Table 1.

Table 1 Parameters for gas chromatographic analysis (GC-FID procedure):

Carrier gas Helium 35 cm min–1

Detector FID; 250 ˚C

Detector gases Hydrogen 45 cm3 min–1

Air 450 cm3 min–1

Injector Split/splitless type; 200 ˚C

Split 10:1

Chromatographic Dikma DM-5 capillary (5% dibenzyl-95% dimethylpolysiloxane)

Column

Length 30 m

Film thickness 0.25 µm

Internal diameter 0.53 mm

Temperature Initial temperature 50 ˚C, ramped at

30 ˚C min–1 to 200 ˚C, hold 3 min; total

analysis time 8 min

Procedure:

Isolation and enrichment of ethanol performed using static headspace technique, while separation performed using gas chromatography with flame ionization detection.

Water with N,N-dimethylacetamide (9:1) was used as dissolving solution.

The sample was incubated in headspace vial (5ml in 20 ml vial) at 80 C˚ for 30 min, 0.5 ml of the vapor phase was injected into the GC-FID system with split mode 10.

The oven temperature was programmed from 50 C˚ to 200 C˚ at 30 C˚/min. 200 C˚ is held for 3 min. The total run time is 8 minutes. Headspace parameters are presented in table 2.

Table 2 Parameters for sample preparation step using headspace technique:

Parameter Value

Thermostat temperature (˚C) 80 ± 2

Needle temperature (˚C) 85 ± 2

Transfer line temperature (˚C) 90 ± 2

Time (min) 30 ± 0.5

Sample amount 5 ml of solution

Injection volume (ml) 0.5

Pressure (kPa) 160 ± 5

Pressurize time (min) 0.5 ± 0.05

Withdraw time (min) 0.2 ± 0.02

Standard solution preparation:

Standard solutions were prepared by adding about 10 ml of water to a 100-ml volumetric flask, followed by 10 ml of N,N-dimethylacetamide and an exact amount of ethanol (using a micropipette). Then the flask was filled up with water to the volume of 100 ml. Five milliliters of each standard solution were transferred to a 20-ml headspace vial and analyzed (HSA-GC-FID). The results of these experiments were plotted as calibration curves over the concentration range from 2500 ppm to 6000 ppm.

Quantitative analysis:

The sample was placed in a 20-ml headspace vial. Then, 5 ml of water with N,N dimethyl acetamide (9:1) were added to the vial. The sample was analyzed using the HS-GC-FID procedure. The chromatographic signal (peak area) and calibration curves of the standard ethanol plotted previously made it possible to estimate the concentration of ethanol. In the sample, once isolation and chromatographic conditions were established, the method validation was performed following ICH recommendations¹⁶.

RESULTS AND DISCUSSION:

The first step of the experimental was the validation which includes the following Parameters: selectivity (specificity), linearity, limit of detection (LOD) and limit of quantitation (LOQ), range, repeatability and intermediate precision.

System suitability test:

Five replications for ethanol standard solution has been injected after that the relative standard deviation was calculated and the calculated value was 2.41%.

Specificity:

The blank sample was injected and analyzed. No significant influence of other compounds on quantification was observed according the basis of the chromatogram obtained. A mixture of methanol and ethanol was prepared and analyzed.

On the basis of this chromatogram there has been good resolution between ethanol and methanol. Rѕ = 1

Linearity:

Linearity was verified by analyzing five standard solutions in the range (2500-6000) ppm of ethanol in the vial, four times each. The relationship between the analyte concentration in the sample and the corresponding detector response was calculated using the linear regression method. On the basis of results obtained, linearity was determined for several concentration ranges. The calibration curve equation was

y=3669351x+3972626 and the correlation factor was 0.995.

Detection limit and quantitation limit:

The blank was injected in the same condition, then the noise was determined then detection limit was calculated from the equation

Detection limit= 3*noise according to United State Pharmacopeia.

The calculated value was LOD= 0.8 ppm. Then quantitation limit was calculated from the equation

LOQ= 10*noise according to United State Pharmacopeia.

The calculated value was LOQ= 2.9 ppm.

Accuracy:

Nine samples has been injected for solutions prepared to estimate accuracy. The concentrations used were (50-80-120) % of the standard solution. Three replications for each concentration. Then the percentage recovery was calculated. The calculated value was 101.06%.

Precision:

Nine samples has been injected for solutions prepared to estimate repeatability. The concentrations used were (50-80-120) % of the standard solution. Three replications for each concentration. Then the percentage recovery was calculated. The calculated value was 99%. In the same way this value for intermediate precision was 98.88%.

Robustness:

The flow rate had been changed in our study (0.4) ml/min increasing and decreasing by 0.1 ml/min to be (0.3-0.4-0.5) in order. Then the five samples of the standard solution had been injected at each flow rate. The relative standard deviation was calculated, the calculated value was less than 15%

Sample analysis:

Sample was incubated with 5 ml of standard ethanol solution 2500ppm in the headspace vial for 30 minutes. Ethanol was analyzed in twelve dosage forms for three drug substances (paracetamol- ibuprofen- amoxicillin) four different tablets for each drug substances.

samples
Paracetamol 500 mg		(Amoxycillin875+ Clavulanate potassium125) mg		Ibuprofen 600mg
1	Panadol gsk	1	Augmentin gsk	1	Brufen Abbott
2	Doprane Oubari	2	Amoxiclav bahri	2	Brufen Unipharma
3	Paracetamol kanawati	3	Clavoxil Elsaad	3	Pofen Elsaad
4	Sytamol Thameo	4	Augmenta Asia	4	Hayabruf Ibn Hayyan

The result obtained was as follow

Paracetamol 500 mg
Sample	Ethanol conc ppm	Result
Cetamol GSK	462.845534	Accepted
Cetamol x	-124.906095	Accepted
Cetamol y	-1145.83159	Accepted
Cetamol z	1724.839835	Rejected

(Amoxycillin875+clavulanate potassium125) mg
Sample	Ethanol conc ppm	Result
Amoxycillin GSK	2535.808241	accepted
Amoxycillin x	-105.4380328	accepted
Amoxycillin y	2351.195224	accepted
Amoxycillin z	998.7023567	accepted

Ibuprofen 600mg
Sample	Ethanol conc ppm	Result
Ibuprofen Abott	874.2414	Accepted
Ibuprofen x	885.64492	Accepted
Ibuprofen y	1703.4348	Accepted
Ibuprofen z	2526.1781	Rejected

CONCLUSIONS:

In this study, a HS-GC-FID analytical method was developed and validated for the qualitative determination of ethanol in a drug substance. Development was carried out according to requirements of the Eur. Ph. General method¹⁷. The validation was performed successfully. The method has been shown to be accurate, linear, precise, reproducible, repeatable, specific, and robust. Sample solvent water with n, n dimethylacet amid (9:1) was selected to obtain good recoveries for ethanol. The conditions of HS sampler and GC were optimized to make the HSGC method more sensitive. Excellent results were obtained within the world wide accepted validation reference values, and particularly taking into account the low concentration levels investigated. This method has been shown to have a high sensitivity since it has a low DL and QL of 0.88 ppm and 2.95 ppm for ethanol. It has also been demonstrated that this method can be readily used to determine ethanol in solid dosage forms. Therefore, this method may also work for other residual solvent analysis. This method has a much shorter sample equilibration time and shorter run time comparing with the previously published methods.

REFERENCES:

1. International Conference on Harmonization (ICH) of Technical Requirement for Registration of Pharmaceutical for Human Use, Topic Q3C, Impurities Guideline for Residual Solvents, 2005, www.ich.org.

2. Jiben Roy, Pharmaceutical Impurities- A Mini-Review, AAPS PharmSciTech 2002, 3 (2) article 6.

3. Saeed Nojavana, Alireza Ghassempourb, Yosef Bashourc, Masoud Khalilian Darbandic, Seyyed Hamid Ahmadid, Determination of residual solvents and investigation of their effect on ampicillin trihydrate crystal structure, Journal of Pharmaceutical and Biomedical Analysis 36 (2005) 983–988.

4. Magdalena Michulec, Piotr Konieczka, Jacek Namies´nik, Validation of the HS-GC-FID method for the determination of ethanol residue in tablets, Accred Qual Assur (2007) 12:257–262.

5. Chang Chenga, Shaorong Liua, Bradford J. Muellerb, Zimeng Yanb, A generic static headspace gas chromatography method for determination of residual solvents in drug substance, Journal of Chromatography A, 1217 (2010) 6413–6421

6. R. S. Lokhande, P. U. Singare, P. V. Jadhav, Development and Validation of Headspace Method for Determination of Residual Solvents in Diphenoxylate Hydrochloride Bulk Drug, American Journal of Chemistry 2012, 2(2): 1-5.

7. Jayalakshmi Somuramasami, Yu-Chien Wei, Emad F. Soliman, Abu M. Rustum, Static headspace gas chromatographic method for the determination of low and high boiling residual solvents in Betamethasone valerate, Journal of Pharmaceutical and Biomedical Analysis 54 (2011) 242–247.

8. Marie-Josée Rocheleau, Mélanie Titley, Julie Bolduc, Measuring residual solvents in pharmaceutical samples using fast gas chromatography techniques, Journal of Chromatography B, 805 (2004) 77–86.

9. Identification and control of residual solvents. In: European Pharmacopoeia 5.0 (2004).

10. B. Kolb, L.S. Ettre, Static Headspace Gas Chromatography—Theory and Practice, Wiley-VCH, New York, 1997.

11. European Pharmacopeia 4th Edition (2.4.24), Identification and Control of Residual Solvents, Council of Europe, Strasbourg, France,2002.

12. United States Pharmacopeia Topic <467>, Organic Volatile Impurities, 2007.

13. Working party “Residual Solvents—Technical” of the European Pharmacopeia Commission, Pharmacopoeia 5 (1993) 145–148.

14. Yingjia Yu1, Bin Chen1, Cidan Shen, Yi Cai, Meifen Xie, Wei Zhou, Yile Chen, Yan Li, Gengli Duan, Multiple headspace single-drop microextraction coupled with gas chromatography for direct determination of residual solvents in solid drug product, Journal of Chromatography A, 1217 (2010) 5158–5164.

15. A.N. Marinichev, A.G. Vitenberg, A.S. Bureiko, J. Chromatogr. 600 (1992) 251.

16. International Conference on Harmonization (ICH) of Technical Requirement for Registration of Pharmaceutical for Human Use, Topic Q2R1, Validation of Analytical Procedures, 2005, www.ich.org.

17. Identification and Control of Residual Solvents (2.4.24), European Pharmacopoeia, Directorate for the Quality of Medicines of the Council of Europe, Strasbourg, fourth ed., 2002, p. 96.

Received on 20.11.2013 Modified on 25.12.2013

Research J. Pharm. and Tech. 7(2): Feb. 2014; Page 184-187