INTRODUCTION:

Since July1, 2008, USFDA has made it mandatory to profile all the residual solvents present in pharmaceutical products and ingredients¹. USFDA urges complete profiling of residual solvents for filing and its approval, since residual solvents had been made official in USP under General Chapters <467>². FDA also recommends controlling the residual solvents in drug products and ingredients which are not described by a USP monograph³.

Acetic acid has been classified as class III residual solvent under ICH guidelines for residual solvents under ICHQ3C (R4); and has a limited permissible daily intake (50 mg/ day)⁴. Acetic acid is one among the aliphatic acids commonly used as excipeints in pharmaceutical products for pH adjustments, providing buffering activity or as solubilization aid⁵. Gelatin also is a common exipient used in pharmaceutical industry for preparing hard and soft gelatin capsule shells⁶. Acetic acid is used as aid for cationic washing, gelatin grafting and pH adjustments during gelatin processing^7,
8. Hence, its presence in finished gelatin shells needs to be monitored.

Gelatin is a collagen product and contains several amino acids and peptides^6,9-11. Further, water soluble and insoluble dyes are added to gelatin to give color identification to shells^6,12-14Presence of all these materials create requirement of a specific and accurate method for determination of acetic acid content in gelatin shells. Some UV methods for acetic acid content determination in gelatin shells are present but utilize several enzymatic redox reactions using NADPH. Direct UV determination of acetic acid cannot be done due to high interference in absorbance window. A variety of gas chromatographic methods are available to determine acetic acid content in different matrixes, but not in gelatin capsule shells¹⁵. Moreover, these techniques are complex and require expertise to conduct. Sensitivity and selectivity are the other problems associated with above methods.

A simple, rapid, accurate and sensitive reversed phase HPLC method was developed to determine the acetic acid content in gelatin matrixes that does not involved any derivatization and engages direct analysis using a substrate.

MATERIALS AND METHODS:

Equipments:

HPLC chromatograph with Waters 2695 Separations Module (Alliance), Waters 2996 Photo Diode Array Detector (Milford, MA, USA) using Empower 2 (database version 6.10.00.00) acquisition software were used in analytical method development. Analytical balance Sartorius Genius (Goettingen, Germany) was used throughout the study for weighing. Millipore Milli-Q Gradient A10 (Billerica, MA, USA), was used for production of HPLC grade water. Julabo TW 12 water bath (CA, USA) was used for sample preparation. All the pH adjustments were done using a Thermo Orion model 410A+ pH meter (Beverly, MA, USA). Hard gelatin capsule shells of different size were procured from Associated Capsules Private Ltd. ((Mumbai, India) and Capsugel (Rewari, India).

Reagents and materials:

To prepare mobile phase and diluent, HPLC grade methanol was procured from Merck (Mumbai, India) and HPLC grade water was produced from Milli-Q Gradient A10 water purification system. Acetic acid working standard (99.9 % pure) was procured from Fluka (Munich, Germany). Hydrochloric acid (AR Grade) and ortho-phosphoric acid (88% w/w AR grade) were procured from Merck (Mumbai, India).

Chromatographic conditions:

Preparation of mobile phase A:

0.7 mL of orthophosphoric acid was diluted upto 1000 mL with water and the pH was adjusted to 3.8 (±0.05) with 10% w/v sodium hydroxide solution in water.

Preparation of mobile phase B:

Methanol was used mobile phase B.

Preparation of diluents:

The pH of water was adjusted to 1.0 (±0.05) with hydrochloric acid. A chromatograph equipped with a Lichrospher RP18e, (250 x 4.0) mm, 5 µm HPLC column maintained at 30°C was used for analysis. The mobile phase flow rate was kept 1.0 mL/min and UV detection was made at 205 nm. 50 µL of the blank, standard and sample solutions were injected. Run times of 6 min for standard solution and 25 minutes for blank and sample solutions were optimized. Acetic acid preparations were stored in ambient conditions. The gradient used was as hereunder-

Time (min)	Mobile Phase A (%)	Mobile Phase B (%)
0	100	0
7	100	0
8	50	50
16	50	50
17	100	0
25	100	0

Standard preparation:

50 µg/ ml of Acetic acid working standard in diluent.

Blank preparation:

Diluent was used as blank preparation.

Sample preparation:

Determine the average weight of intact hard gelatin capsule shells using 20 intact shells. Accurately weigh about 500 mg of hard gelatin capsule shells, separate them in caps and bodies and transfer them all into a 50 ml dried volumetric flask. Add about 40 ml of the diluent in the volumetric flask, shake to disperse the adhered capsule shells, plug the volumetric flask tightly and put in a water bath previously maintained at 40°C for about 20 min with intermittent shaking. Remove the volumetric flask after complete dispersion of contents and cool to room temperature. Unplug the volumetric flask and add 5-10 drops of methanol to remove the frothing (if necessary). Make up the volume with diluent and shake well. Transfer the entire contents of volumetric flask in a 50 ml Tarsons tube and centrifuge at 10000 rpm for 20 min. Chromatograph the supernatant after filtering with 0.45 µm porosity membrane filter.

Evaluation of system suitability:

Prior to analysis, the suitability of the chromatographic system is ensured by injecting blank and standard solutions. Blank is injected to ensure absence of any interference at the retention time of acetic acid. USP tailing (not more than 1.5) and USP plate counts of acetic acid peak in standard solution (not less than 6000) are additionally monitored as system suitability parameters. The system precision of the chromatograph is ensured by checking the % relative standard deviation (% RSD) of area counts of acetic acid peak in six replicate injections of standard solution (not more than 5.0 %). The retention time of acetic acid peak is about 4 minutes.

Calculations:

AT DS P

Acetic acid = ------ x ------ x ----- x 1000000

(µg per gram) AS DT 100

AT DS P

Acetic acid = ------ x ------ x ----- x W x1000000

(µg per capsule shell) AS DT 100

Where; AT = Average area counts for acetic acid peak in the chromatograms of sample solution.

AS = Average area counts for acetic acid peak in the chromatograms of standard solution.

DS = Dilution factor of standard solution.

DT = Dilution factor of sample solution.

W = Average weight of hard gelatin capsule shells

P = Percent purity of acetic acid working standard.

Analytical method development:

HPLC method for Acetic acid determination was initiated as per the method in USP general chapter <503> for determination of acetic acid in peptides. A gradient programme using ortho-phosphoric acid in water, pH 3.8 and methanol as mobile phase components at flow rate of 1 ml min^-1 was evaluated for suitability. Lichrospher RP 18e, 250 x 4.0 mm, 5 micron maintained at 30°C was used as stationary phase. Ultra violet detection was done at 205 nm. A mixture of ortho-phosphoric acid in water, pH 3.0 and methanol in a ratio of 95:5, respectively was used as diluent. A variety of matrix contributed peaks eluted close to acetic acid peak and separation of acetic acid peak with closely eluting peaks was not reproducible.

Table-1: Precision

Hard gelatin capsule shells	Size	Acetic acid content (µg per gram)	Average content (n=3) (µg per gram)	SD	% RSD
Hard Gelatin Capsule Shells Size 1	Size 1, Sample-1	5014.93	5087.17	126.80	2.49
	Size 1, Sample-2	5233.58
	Size 1, Sample-3	5013.01
Hard Gelatin Capsule Shells Size 2	Size 2, Sample-1	6102.59	6148.76	41.14	0.67
	Size 2, Sample-2	6162.17
	Size 2, Sample-3	6181.53
Hard Gelatin Capsule Shells Size 3	Size 3, Sample-1	2988.54	2997.33	63.55	2.12
	Size 3, Sample-2	3064.82
	Size 3, Sample-3	2938.64
Hard Gelatin Capsule Shells Size 4	Size 4, Sample-1	9781.63	9774.04	33.22	0.34
	Size 4, Sample-2	9802.81
	Size 4, Sample-3	9737.69

Thereafter, attempts were made to rectify the separation issues by changing a variety of columns using the above chromatographic conditions (Luna Phenyl Hexyl, 250 x 4.6 mm, 5µm and Atlantis T3, 150 x 4.6 mm, 3µm). Further, chromatographic conditions like column oven temperatures, ion pairing agents, flow rate, HPLC columns (ACE Phenyl, 250 x 4.6 mm, 5µm, Inertsil ODS 3V, 250 x 4.6 mm, 5 micron, ACE C18 HL, 250 x 4.6 mm, 5 µm, ACE 5 AQ, 250 x 4.6 mm, 5 µm) gradient, diluent compositions or components and sample preparation techniques were modified, but a suitable conditions specific for acetic acid determination could not be achieved. Later the sample preparation was optimized in hydrochloric acid solution (pH adjusted to 1.0) and the analysis was done on Lichrospher RP 18e, 250 x 4.0 mm, 5 micron. Conditions were found to be specific and reproducible. The chromatographic conditions optimized were evaluated for their suitability and applicability as per ICH guidelines. The various parameters evaluated were as hereunder-

Table-2: Accuracy

Hard gelatin capsule shells	Size	% Recovery	Mean of % recovery (n=3)
Hard Gelatin Capsule Shells Size 1	Size 1, Sample-1	102.97	102.49
	Size 1, Sample-2	100.96
	Size 1, Sample-3	103.54
Hard Gelatin Capsule Shells Size 2	Size 2, Sample-1	98.87	97.67
	Size 2, Sample-2	96.27
	Size 2, Sample-3	97.87
Hard Gelatin Capsule Shells Size 3	Size 3, Sample-1	104.55	103.7
	Size 3, Sample-2	104.26
	Size 3, Sample-3	102.29
Hard Gelatin Capsule Shells Size 4	Size 4, Sample-1	100.31	101.29
	Size 4, Sample-2	100.99
	Size 4, Sample-3	102.58

Fig 1: Blank Chromatogram

RESULTS AND DISCUSSIONS:

Specificity:

Blank was injected as part of system suitability. No interference was observed at the retention time of acetic acid. The peak purity of acetic acid passed in sample preparations. (Fig. 1 and 2)

Fig 2: Sample Chromatogram

Precision:

Samples from both the vendors for size- 0, 1, 3 and 4 were prepared in triplicate and analyzed for acetic acid content (Table-1)

Accuracy:

Recovery was done in triplicate by spiking acetic acid in pre-analyzed hard gelatin capsule shells from both the vendors (for size-0, 1, 3 and 4) at 50 % and 100 % level of specification limit (5000 ppm) (Table-2).

Stability in analytical solution (SIAS):

Stability of acetic acid in analytical solution was determined at ambient storage, both in the standard as well as in the sample preparation. Acetic acid was found stable for not less than 14 hrs in both the preparations (Table-3).

Table 3: Stability in analytical solution

Time (min)	Acetic acid in standard solution		Acetic acid in sample preparation
	Area	% Cumulative RSD	Area	% Cumulative RSD
Initial	102944	-	66339	-
123	103017	0.05	65547	0.85
246	102164	0.46	65208	0.88
369	102677	0.38	63496	1.84
492	102420	0.35	63574	1.93
614	103004	0.34	63827	1.85
737	102136	0.38	63805	1.76
860	102250	0.37	64017	1.66

Limit of quantification (LOQ):

A LOQ of ~ 2.5 ppm was achieved with a % RSD below 5 % (Table-4).

Table 4: LOQ

Concentration of acetic acid in LOQ preparation: 2.5 µg/ ml
Injection	Area	Signal to noise ratio
1	4792	10.903
2	4332	5.047
3	4890	9.657
4	4455	7.450
5	4597	6.146
6	4867	5.538
Mean	4656
% RSD	4.96

REFERENCES:

1. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm073394.pdf , Accessed on 15 June, 2010.

2. USP 32 NF 27, 2009, Chapter <467> Residual Solvents, The USP Convention, Rockville, MD, 163.

3. http://www.usp.org/USPNF/notices/generalChapter467.html , Accessed on 15 June, 2010.

4. http://www.ich.org/LOB/media/MEDIA5254.pdf, Accessed on 15 June, 2010.

5. Chambliss WG. Acetic acid, Glacial. In Encyclopedia of Pharmaceutical Exipients, Edited by Raymond CR, Paul JS and Sian CO. Pharmaceutical Press, London, UK. 2006; 5th ed: pp. 6-7.

6. Price JC. Gelatin. In Encyclopedia of Pharmaceutical Exipients, Edited by Raymond CR, Paul JS and Sian CO. Pharmaceutical Press, London, UK. 2006; 5th ed: pp. 295-298.

7. George A, Radhakrishnan G and Joseph KT. Modification of gelatin by grafting. Die Angewandte Makromolekulare Chemie. 2003; 131: 169-176.

8. Northrop JH and Kunitz M. Preparation of electrolyte-free gelatin. J Gen Physiol. 1928; 11: 477-479.

9. Williams AP. The chemical composition of snail gelatin. Biochem J. 1960; 74: 304-307.

10. http://www.gmap-gelatin.com/about_gelatin_comp.html , Accessed on 21 May, 2010.

11. http://www.gelatin-gmia.com/html/gelatine_health.html , Accessed on 21 May, 2010.

12. http://www.aapsj.org/abstracts/AM_2006/staged/AAPS2006-001511.PDF , Accessed on 21 May, 2010.

13. http://www.erawat.com/empty-hard-gelatin-capsules.html , Accessed on 21 May, 2010.

14. http://www.capsugel.com/services/regulatory_colorants.php , Accessed on 21 May, 2010.

15. Yang MH and Choong YM. A rapid gas chromatographic method for direct determination of short-chain (C₂–C₁₂) volatile organic acids in foods. Food chemistry. 2001; 75: 101-108.

Received on 29.06.2010 Modified on 17.07.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 1058-1061