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Simultaneous Estimation of Menthol, Camphor and Methyl Salicylate in Anti-Arthritic Spray Using Gas Chromatography

Munshi Mahammed Tanzil Mahammed Aslam1*, Saisivam Srinivasan1, Rabadiya Vibhuti Amitkumar1, Shailendra K. Mehta2

1 Department of Pharmacy, N. R. Vekaria Institute of Pharmacy, Gujarat Technological University, Junagadh, Gujarat, India

2 ICPA Healthcare Products Ltd. Ankleshwar, Gujarat, India

Munshi Mahammed Tanzil Mahammed Aslam
Department of Pharmacy,
N. R. Vekaria Institute of Pharmacy,
Gujarat Technological University,
Junagadh,
Gujarat,
India
E-mail: [email protected]

Received: 11-Feb-2022, Manuscript No. JPA-22-53967; Editor assigned: 14-Feb-2022, PreQC No. JPA-22-53967(PQ); Reviewed: 25-Feb-2022, QC No. JPA-22-53967; Revised: 28-Feb-2022, Manuscript No. JPA-22-53967(R); Published: 07-Mar-2022, DOI:10.4172/2320-0812.11.02.002

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Abstract

A simple, rapid, accurate, precise and robust Gas Chromatographic (GC) method has been developed for the simultaneous estimation of Menthol, Camphor and Methyl Salicylate from an anti-arthritic spray. The separation of these three constituents was performed on DB-23 column (60 m × 250 µm × 0.25 µm). The Nitrogen was used as carrier gas at a flow rate of 2.5 mL/min. The inlet temperature and detector temperature were set as 250°C and 280°C respectively. The initial oven temperatures was 125°C for 5 min, then increase to 150°C at the ramp of 3°C/min and finally reach to 200°C at the ramp of 10°C/min. The ALS (Automatic Liquid Sampler) system was used for sample injection. The injection volume was 2 µl. The run time of chromatogram was 20.333 min. The proposed GC method provides a good resolution of Menthol, Camphor and Methyl Salicylate. The method was validated as per ICH guidelines. The linearity of Menthol, Camphor and Methyl Salicylate were found to be in the range of 10-50 µg/mL, 20-60 µg/mL and 100-300 µg/mL respectively. The recovery was calculated by using external standard method and the mean average recovery for Menthol, Camphor and Methyl Salicylate were found to be 100.55%, 99.93% and 99.95%. The tested validation parameters were found to be within acceptable limits. The method was successfully applied for quantification of these three ingredients in anti-arthritic spray formulation.

Keywords

Gas chromatography; Active ingredients; Automatic liquid sampler; Anti-arthritic spray; Inflammation

Introduction

Menthol, Camphor and Methyl Salicylate are the active ingredients of many topical formulations used for the treatment of rheumatic diseases. These are common ingredients used in certain topical preparation due to their analgesic and anti-inflammatory activity [1] (Figure 1).

pharmaceutical-analysis-ingredients

Figure 1: Structures of active ingredients.

Since time immemorial, various herbs and their derived compounds have been used in treatment of inflammation. Inflammation is a normal, protective response to tissue injury caused by physical trauma, noxious chemicals or microbiologic agents, which is a part of the host defense. Sometimes, inflammation seems to produce events that are quite serious and become chronic like occurrence of rheumatoid arthritis and hay fever which may be life threatening. Rubor (redness), calor (heat), tumor (swelling) and dolor (pain) are the main signs of inflammation [2].

S. P. Sethy has found from his review that GC with capillary column can be suitable for simultaneous identification and detection of active substance as well as purity evaluation in the presence of matrix constituents. Thus such an injection method increase the stability of the injected sample, thus also decreasing the number of peaks in the chromatogram Therefore an attempt has been made to establish conditions for identification and determination active constituents in the presence of a base constituents, while considering the effect on the results obtained from an gel containing menthol and methyl salicylate by using capillary gas chromatography [3].

Because of the volatility of these constituents, it is not surprising that these compounds are generally determined with GC. Few reports can be found about the determination of these constituents in pharmaceutical products [4].

A recent trip to a community pharmacy revealed 35 different ingredients. Of those products, 16 contained only menthol in amount ranging from 1.27% to 16%. One spray product listed menthol as the only active ingredients but no content was given. Two products contained menthol and camphor only, but neither listed content for camphor and only one product listed menthol at 2.5%. Fourteen products contained menthol and 30% methyl salicylate. One of the store brand product contained menthol and methyl salicylate as active ingredients in unlisted amounts. Three products contained all the ingredients, with the most concentrated product containing 30% methyl salicylate, 10% menthol and 4% camphor. These products are manufactured and sold in a variety of forms including: creams, gels, balms, rub-on-sticks, patches, pain reliever mousses etc [5].

Assay methods for the quantitative determination of combinations of any two of these drugs have been reported. These methods were based on Gas-Liquid Chromatography (GLC) since these drugs are volatile substances. However, only a few methods were published which allowed the assay of mixtures containing all three drugs [6].

Since many pharmaceutical preparations contain camphor and menthol, it is desirable to use a method which is both fast and accurate for the simultaneous determination of camphor and menthol [7].

In this paper, we have reported a gas chromatographic method for simultaneous estimation of these three constituents from anti-arthritic spray which contains Menthol, Camphor and Methyl Salicylate 3.0%, 4.0% and 20% respectively. The literature review reveals that till now, no any published method is reported for estimation of these three constituents from anti-arthritic spray which is research product yet to be commercialized. To confirm suitability of developed method for routine analysis, method was validated as per the ICH guidelines [8].This method can be successfully applied for routine quality control analysis.

Materials and Methods

The method was developed on GC of Agilent Technologies equipped with FID (Flame Ionization Detector) in 2017-18 at ICPA healthcare products ltd. Ankleshwar by using following parameters.

Chromatographic parameters

• Column: DB-23 (60 m × 250 μm × 0.25 μm)

• Oven temperature: 125°C(5 min), increase to 150°C (ramp 3°C/min), reach to 200°C (ramp 10°C/min)

• Time of run: 20.333 min

• Inlet temperature: 250°C

• Detector temperature: 280°C

• Mode: Split

• Split ratio: 20:

• Injection volume: 2 μl

• Carrier gas: Nitrogen

• Flow rate: 2.5 mL/min

• H2 flow: 30 mL/min

• Air flow: 300 mL/min

• N2 makeup flow: 25 mL/min

Reagents and materials

The following reagents and materials were used: Menthol of Hindustan Mint and Agro Products Pct. Ltd., Camphor of Camphor and Allied Product, Methyl salicylate of John Aromas and Methanol of Merck (all ingredients available at ICPA healthcare product ltd. Ankleshwar, Gujarat, India).

Preparation of standard mixture

Accurately weighed quantity of 1 gm Menthol, 1 gm Camphor and 4 gm Methyl Salicylate were transferred into separate 100 mL volumetric flasks; dissolved each in 50 mL of methanol and sonicated for 15 minutes. Final volume was made upto 100 mL with methanol of each flask to prepare standard stock solutions having 10000 μg/mL Menthol, 10000 μg/mL Camphor and 40000 μg/mL Methyl Salicylate concentrations. From those stock solutions, 6 mL of Menthol, 8 mL of Camphor and 10 mL of Methyl Salicylate were withdrawn from respective flasks and transferred into one common 100 mL volumetric flask and final volume was made up with methanol.

Preparation of test solution

The anti-arthritic spray sample was shaken well and 2 mL was withdrawn and transferred to 100 mL volumetric flask; dissolved in 50 mL of methanol and sonicated for 20 minutes. Final volume was made up to 100 mL with methanol.

Chromatographic procedure

Using ALS (Automatic Liquid Sampler) equipped with 10 µl syringe, 2 µl of standard solution and 2 µl of test solution were injected into gas chromatographic system under the operating conditions described above and chromatograms were recorded (Figures 2 and 3).

pharmaceutical-analysis-mixture

Figure 2: Standard mixture.

pharmaceutical-analysis-sample

Figure 3: Test sample.

Assay

The individual solutions of Menthol (600 ppm), Camphor (800 ppm) and Methyl Salicylate (4000 ppm) were injected to the GC system with respect to developed chromatographic method and then sample solution was injected (Table 1). The area of the standard solutions as well as sample was determined and assay was performed by using following equation:

equation

Method validation

Specificity: Specificity is a procedure to detect quantitatively the analytes of interest in presence of components that may be present in the sample matrix. Specificity was tested against standard compounds and in the formulation against potential interferences of excipients.

Linearity and range: The linearity is evaluated through a linear regression analysis. Linearity levels for Menthol, Camphor and Methyl Salicylate at range of 10-50 µg/mL, 20-60 µg/mL and 100-300 µg/mL respectively (as per their concentrations in formulation) were determined in terms of correlation coefficient (Figures 4-6) (Tables 2-4).

Accuracy: The analytical method should be able to recover actual amount of the analyte from the formulation. Accuracy was determined by calculating the %recovery by External Standard method in which three Placebo samples were taken and known amount of Menthol, Camphor and Methyl Salicylate were added in them at concentrations of 80%, 100% and 120% respectively. These solutions were injected in GC system and %Recovery was calculated. These were repeated 3 times and mean recovery was calculated (Tables 5-7).

Precision: Precision was considered at different levels.

Repeatability: Repeatability expresses the precision under same operating conditions over short interval of time. Take 0.1 mL of test solution in 100 mL volumetric flask and diluted up to mark with Methanol to get concentrations of 30 μg/mL Menthol, 40 μg/mL Camphor and 200 μg/mL Methyl Salicylate and analyze the procedure for 10 times and get the result (Table 8).

Intermediate precision: Intermediate precision was determined by analyzing the test solution (0.1 mL test sample in 100 mL Methanol) 10 times by different analysts and by same analyst in different days (day 1 and day 8) and %RSD was calculated (Tables 9 and 10).

Intraday precision: Intraday precision was determined by analyzing the test solution (0.1 mL test sample in 100 mL Methanol) 10 times in the same day and %RSD was calculated (Tables 11 and 12).

Robustness: The robustness of the method was established by making deliberate minor variations in the method parameters (Tables 13-15).

Change following parameters one by one and observe their effect on assay.

i. Change in mobile phase flow rate by ± 0.2 mL/min

ii. Change in initial oven temperature by ± 5 °C

System suitability: System suitability testing is an integral part of many analytical procedures. System suitability test parameters to be established for a particular procedure depend on the type of procedure being validated. Here, Resolution between all three peaks, Theoretical plates and Peak Asymmetry were calculated by injecting test samples 5 times and % RSD was calculated (Tables 16-18).

Results and Discussion

Assay

Constituents % Assay
Menthol 99.97%
Camphor 98.54%
Methyl Salicylate 99.56%

Table 1. Assay of developed formulation.

Specificity

No interference was detected at the retention time of Menthol, Camphor and Methyl Salicylate in sample solution. 

Linearity and range

pharmaceutical-analysis-menthol

Figure 4: Linearity of menthol.

Menthol
Concentration Peak Area
0 0
10 33548
20 61339
30 89669
40 117476
50 144503

Table 2. Linearity and range of menthol.

pharmaceutical-analysis-camphor

Figure 5: Linearity of camphor.

Camphor
Concentration Peak Area
0 0
20 56922
30 89223
40 118385
50 144700
60 174320

Table 3. Linearity and range of camphor.

pharmaceutical-analysis-methyl

Figure 6: Linearity of methyl salicylate.

Methyl Salicylate
Concentration Peak Area
0 0
100 199567
150 305570
200 398666
250 506184
300 600486
 

Table 4. Linearity and range of methyl salicylate.

Accuracy

Menthol
Sr. No. % Level % of menthol added in placebo % Assay % RSD % Recovery Average % Recovery ± SD
1 80 81.35 80.89 0.4247 101.11 101.07 ± 0.1923
2 80.84 80.65 100.81
3 81.19 81.02 101.28
1 100 100.40 100.78 0.4587 100.78 100.09 ± 0.4452
2 100.69 99.43 99.43
3 100.28 100.07 100.07
1 120 121.00 120.62 0.3013 100.51 100.50 ± 0.4558
2 120.74 121.26 101.05
3 121.20 119.92 99.93
Mean % Average Assay 100.55 ± 0.4007

Table 5. Recovery study of menthol.

Camphor
Sr. No. % Level % of camphor added in placebo % Assay % RSD % Recovery Average % Recovery ± SD
1 80 80.58 80.14 0.6126 100.17 99.85 ± 0.6042
2 80.75 79.20 99.00
3 80.84 80.29 100.37
1 100 100.32 99.91 0.5126 99.91 99.69 ± 0.4602
2 100.25 100.12 100.12
3 100.16 99.05 99.05
1 120 120.24 120.13 0.1788 100.11 100.24 ± 0.1481
2 120.28 120.20 100.16
3 120.34 120.54 100.45
Mean % Average Assay 99.93 ± 0.4041

Table 6. Recovery study of camphor.

Methyl Salicylate
Sr. No. % Level % of camphor added in placebo % Assay %RSD % Recovery Average % Recovery ± SD
1 80 80.05 80.16 0.4696 100.20 99.68 ± 0.4849
2 80.08 79.23 99.03
3 80.07 79.84 99.80
1 100 100.06 99.92 0.3464 99.92 100.34 ± 0.3358
2 100.07 100.35 100.35
3 100.08 100.74 100.74
1 120 120.09 119.67 0.2747 99.73 99.82 ± 0.2838
2 120.10 119.44 99.53
3 120.07 120.25 100.21
Mean % Average Assay 99.95 ± 0.3681

Table 7. Recovery study of methyl salicylate.

Precision

Repeatability

Sr. no. Menthol Camphor Methyl salicylate
RT (min) Peak area RT (min) Peak area RT (min) Peak area
1 6.867 89651 7.362 117294 9.355 397922
2 6.878 88852 7.36 118485 9.362 398568
3 6.861 89769 7.366 118395 9.351 397025
4 6.861 89689 7.372 119285 9.354 398190
5 6.869 89702 7.365 117385 9.361 398126
6 6.864 89569 7.369 118402 9.36 397590
7 6.873 88669 7.362 118345 9.359 399489
8 6.865 89675 7.364 116585 9.36 389012
9 6.871 88969 7.37 118485 9.355 398663
10 6.868 90193 7.365 119385 9.356 389564
Mean 6.8677 89473.8 7.3655 118204.6 9.3573 396414.9
SD 0.00508 455.2893 0.00364 832.8762 0.003407 3619.129
%RSD 0.0739 0.5088 0.0494 0.7046 0.0364 0.9129

Table 8. Repeatability

Intermediate precision

Day 1 of interday precision and analyst 1 precision are considered to be same as repeatability.

DAY–VIII, ANALYST-I
Sr. no. Menthol Camphor Methyl salicylate
RT (min) Peak area RT (min) Peak area RT (min) Peak area
1 6.865 89640 7.360 117405 9.354 398129
2 6.877 88765 7.361 118398 9.362 398568
3 6.881 88799 7.366 118612 9.351 397025
4 6.879 89486 7.372 119081 9.355 398190
5 6.864 89512 7.385 117854 9.371 398126
6 6.865 88956 7.369 118366 9.360 397590
7 6.870 89570 7.362 117447 9.349 399489
8 6.866 87999 7.364 118476 9.345 389012
9 6.876 88406 7.370 118333 9.355 398663
10 6.869 89454 7.365 118994 9.356 389564
Mean 6.8712 89058.7 7.3674 118296.6 9.3558 396435.6
SD 0.006129 534.4996 0.00696 544.2794 0.006911 3628.27
%RSD 0.0891 0.6001 0.0944 0.4600 0.0738 0.9152

Table 9. Interday precision at day 8.

ANALYST-II
Sr. no. Menthol Camphor Methyl salicylate
RT (min) Peak area RT (min) Peak area RT (min) Peak area
1 6.860 90177 7.372 117899 9.364 398875
2 6.864 89635 7.368 117885 9.356 398112
3 6.859 89225 7.370 119045 9.365 389898
4 6.861 90368 7.371 117466 9.360 398853
5 6.861 88996 7.378 119154 9.359 398213
6 6.864 89235 7.369 117980 9.358 397562
7 6.865 89534 7.368 118036 9.365 395633
8 6.860 89662 7.370 119256 9.359 398863
9 6.863 89457 7.361 118622 9.368 386900
10 6.862 90111 7.368 118765 9.368 398876
Mean 6.862 89640.0 7.370 118410.8 9.362 396178.500
SD 0.0019 428.5340 0.0040 599.6747 0.0041 4056.2496
%RSD 0.0280 0.4781 0.0544 0.5064 0.0437 1.0238

Table 10. Intermediate precision by analyst 2.

Intraday precision

DAY–I, ANALYSIS-I
Sr. no. Menthol Camphor Methyl salicylate
RT (min) Peak area RT (min) Peak area RT (min) Peak area
1 6.861 89888 7.369 118645 9.361 398850
2 6.862 88354 7.368 117840 9.359 389523
3 6.861 88569 7.370 118752 9.361 397564
4 6.870 89567 7.366 116662 9.357 398047
5 6.871 89043 7.368 118003 9.362 398856
6 6.869 90111 7.363 118753 9.367 398740
7 6.872 88045 7.370 119874 9.359 398500
8 6.870 90256 7.372 118630 9.355 398014
9 6.861 89114 7.367 118554 9.368 386483
10 6.865 88236 7.368 118976 9.357 389711
Mean 6.866 89118.3 7.368 118468.9 9.361 395428.800
SD 0.0044 767.8271 0.0023 795.8635 0.0040 4577.5865
%RSD 0.0641 0.8616 0.0318 0.6718 0.0428 1.1576

Table 11. Intraday precision analysis 1.

DAY-I, ANALYSIS-II
Sr. no. Menthol Camphor Methyl salicylate
RT (min) Peak area RT (min) Peak area RT (min) Peak area
1 6.868 89874 7.364 117459 9.359 397985
2 6.862 88463 7.369 117946 9.358 398457
3 6.860 89156 7.366 119350 9.360 398024
4 6.859 88687 7.368 118868 9.362 397843
5 6.865 90041 7.370 118705 9.361 397825
6 6.864 89337 7.365 117985 9.357 398051
7 6.868 89024 7.364 117563 9.368 389921
8 6.866 88647 7.368 118912 9.365 397853
9 6.862 90178 7.366 118175 9.358 386631
10 6.861 89630 7.372 119045 9.362 395530
Mean 6.864 89303.7 7.367 118400.8 9.361 395812.000
SD 0.0030 579.5888 0.0025 625.1182 0.0033 3912.0454
%RSD 0.0443 0.6490 0.0342 0.5280 0.0348 0.9884

Table 12. Intraday precision analysis 2.

Robustness

Robustness of menthol

Condition % RSD of MEN in Std mixture % RSD of MEN in test % Average Assay %Difference in Assay
(1) Change in flow rate ± 0.2 mL/min
2.5 mL/min (Normal) 0.3357 0.6964 100.08 -
-0.2(2.3 mL/min) 0.5095 0.3778 100.14 0.06
+0.2(2.7 mL/min) 0.1962 0.2887 100.59 0.51
(2) Change in initial oven temperature 5 °C
125 °C (Normal) 0.3357 0.6964 100.08 -
-5(120 °C) 0.1533 0.3829 99.98 0.1
+5(130 °C) 0.4570 0.1546 100.19 0.11

Table 13. Robustness of menthol.

Robustness of camphor

Condition % RSD of CAM in Std mixture % RSD of CAM in test % Average Assay % Difference in Assay
(1) Change in flow rate ± 0.2 mL/min
2.5 mL/min (Normal) 0.2608 0.2620 100.78 -
-0.2(2.3 mL/min) 0.2609 0.3405 99.99 0.79
+0.2(2.7 mL/min) 0.5106 0.5638 100.14 0.64
(2) Change in initial oven temperature 5 °C
125 °C    (Normal) 0.2608 0.2620 100.78 -
-5(120 °C) 0.8100 1.2301 100.00 0.78
+5(130 °C) 0.8271 0.7622 100.70 0.08

Table 14. Robustness of camphor.

Robustness of methyl salicylate

Condition % RSD of MeS in Std mixture % RSD of MeS in test % Average Assay % Difference in Assay
(1) Change in flow rate ± 0.2 mL/min
2.5 mL/min (Normal) 0.7621 0.7526 100.08 -
-0.2(2.3 mL/min) 1.1732 1.1506 100.44 0.36
+0.2(2.7 mL/min) 0.6571 0.3179 101.04 0.96
(2) Change in initial oven temperature 5 °C
125 °C(Normal) 0.7621 0.7526 100.08 -
-5(120 °C) 0.6869 0.8176 99.46 0.62
+5(130 °C) 1.0876 0.3588 99.72 0.36

Table 15. Robustness of methyl salicylate.

System suitability parameters

Sr. no. Menthol
Resolution Theoretical plates Asymmetry
1 0 68054 1.10288
2 0 65986 1.09914
3 0 67843 1.09874
4 0 69331 1.10341
5 0 68877 1.09965
Mean 0 68018.200 1.101
SD 0 1150.8949 0.0020
%RSD 0 1.6920 0.1792
Limit >2 >2000 <2

Table 16. System suitability parameters for menthol.

Sr. no. Camphor
Resolution Theoretical plates Asymmetry
1 4.78 87571 1.00019
2 4.79 89631 0.99865
3 4.77 88014 1.00547
4 4.79 85122 1.01372
5 4.78 87103 0.99971
Mean 4.782 87488.2 1.003548
SD 0.0075 1457.5455 0.0056
%RSD 0.1565 1.6660 0.5587
Limit >2 >2000 <2

Table 17. System suitability parameters for camphor.

Sr. no. Methyl Salicylate
Resolution Theoretical plates Asymmetry
1 20.11 147311 0.87564
2 20.09 150744 0.90411
3 20.10 146682 0.89772
4 20.11 148134 0.89980
5 20.10 152365 0.91003
Mean 20.102 149047.2 0.89746
SD 0.0075 2160.0659 0.0117
%RSD 0.0372 1.4492 1.3031
Limit >2 >2000 <2

Table 18. System suitability parameters for methyl salicylate.

Conclusion

A simple, accurate, rapid and economic Gas Chromatographic method has been developed and validated for simultaneous estimation of Menthol, Camphor and Methyl Salicylate in developed anti-arthritic spray formulation. The method assured satisfactory linearity, accuracy and precision. The method is robust and % recovery in spray formulation is good. Analysis of sample containing Menthol, Camphor and Methyl Salicylate showed no interference from the other excipients and additives. The proposed method can be easily and conveniently adopted for routine quality control analysis.

Acknowledgement

• We hereby acknowledge Mr. H. M. Panchal, Dr. Atul Bedekar, Mr. Vinod Patanwala, Mr. Aniruddh Patil, Mr. Mangeshkumar Padame, Mr. Sandesh Shah, Mrs. Sheetal Rajurkar, Mr. Virendra Shah of ICPA Healthcare Products ltd. Ankleshwar, Gujarat for their timely help and knowledge sharing.

• We kindly acknowledge Shree Nanjibhai Vekaria for providing the facilities to carry out this research work.

References

http://www.encyclopaediaislamica.com/