Abstract
A precise, sensitive, specific and accurate stability indicating densitometric method was developed and validated for alpha-lipoic acid (ALA) in bulk and capsule dosage form. The study employed pre-coated silica gel 60F254 TLC plates as stationary phase and toluene: chloroform: methanol: formic acid (5:3:1:0.05; v/v/v/v) as mobile phase. The developed method furnished compact spots of alpha-lipoic acid (Rf 0.28 ± 0.05) after derivatization, offered good linearity in range 80–400 ng/spot with correlation coefficient of 0.998. The values for detection and quantitation were found 18.022 and 54.612 ng/spot respectively. ALA was subjected to stress degradation studies and total 13 degradation products were resolved. Thus, the proposed method offered good results according to ICH guidelines, and can be used for identification, routine quantitative determination as well as for monitoring the stability of ALA in bulk and in capsules.
Introduction
Chemical stability of pharmaceutical molecules is a matter of great concern as it affects the drug safety and efficacy. Stability-indicating methods are quantitative analytical methods that distinguish each active ingredient from its degradants, measuring the content of active ingredient accurately [1, 2].
Alpha-lipoic acid (ALA), also known as thioctic acid, was discovered in 1951 as a molecule with potent antioxidant activity which assists in transfer of acyl-group and acts as a coenzyme in Krebs' cycle [3, 4]. It is a caprylic acid-derived organosulfur compound. Chemically, it is 1,2-dithiolane- 3-pentanoic acid (Fig. 1), which inhibits oxidative stress in cells [4, 5]. It is yellowish powder, poorly water soluble but easily soluble in methanol, acetonitrile, and chloroform [6]. ALA is extensively used in food supplements due to its antioxidant properties and finds wide clinical applicability in the management of many ailments such as diabetes mellitus, hypertension, Alzheimer's disease, Down syndrome, cognitive dysfunction, and breast cancer [7, 8]. As a therapeutic and nutritional supplement, the use of ALA is growing at a very fast pace [9, 10].
High-performance thin-layer chromatography (HPTLC) has emerged as an important tool in the analysis of various drugs and their formulations. Due to several key benefits, such as low running costs, high resolution of complex mixtures, high precision and accuracy, it has surpassed other chromatographic techniques [11, 12].
Literature survey revealed different methods for ALA estimation, either alone or in combination with other drugs, such as UV spectrophotometric method [13], stability assay of ALA and enzogenol by ultra-performance liquid chromatography (UPLC) [14], quantitation and stability of ALA in cosmetic creams by high-performance liquid chromatography (HPLC) [15] evaluation of thermal and photo stability of ALA in encapsulated form [16] quantification of ALA with pulse amperometric detection using HPLC [17]. In combination, ALA has been estimated with gabapentin and mecobalamin by HPLC [18], with low-molecular-mass thiols by HPLC [19] docetaxel by HPLC [20] pregabalin and mecobalamin by reverse phase HPLC [21] metformin hydrochloride and ALA by high-performance thin-layer chromatography (HPTLC) [22]. To the best of our knowledge, stability indicating HPTLC method of ALA alone, in bulk and capsule dosage form through derivatization has not been reported so far, therefore it was thought worthwhile to develop a validated, specific, precise and accurate stability indicating HPTLC method for ALA in bulk and capsule, according to ICH guidelines [23, 24].
Experimental
Instrumentation
The CAMAG HPTLC system consisted of ATS 4 autosampler with microliter syringe (CAMAG, Switzerland), a twin trough chamber (20 × 10 cm), CAMAG derivatizer, and TLC plate heater. Pre-coated silica gel 60 F254, aluminium plates (20 × 10 cm with 200 µm thickness; Merck, Germany) were used as stationary phase. Densitogram scanning was performed by CAMAG Scanner-4.
Other instruments used in study were Digital analytical balance: AUX 220 (Shimadzu, Kyoto, Japan), melting point apparatus: VMP-DS (Veego instruments, Mumbai, India), UV double-beam spectrophotometer: UV-1800 (Shimadzu) and hot air oven: NOVA Instruments (Ahmadabad, India).
Solvents and reagents
ALA was received from Maxtar Bio-Genics Solan, H.P, India. INLIFE TM ALPHA LIPOIC ACID 300 mg capsules were purchased from a pharmacy retail store. Analytical grade solvents viz toluene, chloroform, methanol, formic acid and phosphomolybdic acid were purchased from Merck -Millipore (Mumbai, India).
Standard solution
It was prepared by accurately weighing ALA (10 mg) and dissolving in 10 mL methanol (corresponding to 1,000 μg mL−1), further dilution was done using methanol (100 μg mL−1). Using ATS 4 autosampler, 80–400 ng/spot were applied on pre-coated TLC plates.
Sample solution
The average weight of 20 capsules of INLIFE TM was calculated and contents were finely powdered. Quantity equivalent to 10 mg ALA was dissolved in 10 mL methanol and centrifuged at 2000 rpm for 5 min. The supernatant was applied on TLC plate by using ATS 4 autosampler to get concentration of 200 ng/spot.
Chromatographic conditions
The prepared samples of drug and its marketed formulation were spotted as 8 mm bands on pre-coated silica gel 60 F254, aluminium plate using CAMAG ATS 4 autosampler consisting of CAMAG microliter syringe (Switzerland). Methanol was employed for prewashing the plates followed by activation for 5 min before application of samples. Twin trough glass chamber (20 × 10 cm) was saturated with prepared mobile phase of toluene: chloroform: methanol: formic acid (5:3:1:0.05; v/v/v/v; pH 2.2) by using saturation pads for 20 min. Migration time was maintained as 15 min covering the migration distance of 7 cm. For visualization of spots derivatization was carried out by using phosphomolybdic acid solution (10 g in 50 mL of 96% ethanol). After derivatization, TLC plate was heated at 120 °C for 10 min. Images were captured in white light. The plate was scanned at 600 nm by using CAMAG Scanner-4 and visualized using vision CATS version 2.5.18262.1 software.
Method validation
Linearity and range
Five spots of standard solution of the drug (80–400 ng/spot) were applied on pre-coted plate for development and then analysed to evaluate linearity. The calibration-curve was plotted for peak area vs drug concentration using visionCATS software and analysis of linear regression was carried out.
LOD and LOQ
Accuracy studies
It was expressed as percentage recovery, and was performed by spiking 80%, 100% and 120% of standard drug to the formulation, using standard addition method in triplicates.
Precision studies
Three replicates of ALA concentration (200 ng/spot) were applied and analysed for intra and inter-day precision.
Specificity
Specificity of a method provides an accurate and precise estimation of a targeted analyte in presence of other components in sample matrix. It was performed to evaluate the possibility of interference of impurities, degradants and formulation excipients.
Assay
To determine the amount of ALA in INLIFETM capsule, (label claim: 300 mg ALA per capsule), twenty capsules were opened, contents were weighed and mixed. Powder (equivalent to 10 mg) was weighed, dissolved in 10 mL methanol (1 mg mL−1 concentration), further dilution was done using methanol.
Degradation study
Acid and alkaline degradation
For acid-induced degradation, ALA (10 mg) was dissolved in 10 mL, 0.1N methanolic solution of hydrochloric acid and refluxed at 60°C for 45 min in the dark to avoid interference of light. For alkaline degradation, drug concentration (1 mg mL−1) was prepared using 0.1N methanolic NaOH solution and was refluxed in dark at 60°C for 45 min.
Oxidative degradation
In this study 10 mL of hydrogen peroxide (6% v/v) was added to drug solution of ALA having concentration 1 mg mL−1 and was kept for 45 min in dark at room temperature.
Photochemical degradation
For photochemical degradation, ALA was exposed to direct sunlight for 8 h daily for six days corresponding to 48 h.
Thermal degradation
ALA (10 mg) was kept in oven for 4h at 60°C, and then solution of 1 mg mL−1 was prepared with methanol.
Result and discussion
Method-development and validation
A densitometric method, using toluene: chloroform: methanol: formic acid (5:3:1:0.05, v/v/v/v, pH 2.2) as mobile phase, was developed and scanned after derivatizing it with phosphomolybdic acid. A sharp peak of ALA was observed at Rf 0.28 ± 0.05 (Fig. 2). For validation ICH Q2(R1) guidelines were followed [24].
Linearity
Calibration curve of ALA exhibited a linear relationship between area of peak and concentration in range of 80–400 ng/spot (five data points) as shown (Figs 3 and 4). The regression data of graph was found linear with best correlation r 2 ≥ 0.998 (Table 1).
3D densitogram of 80,160, 240, 320 and 400 ng/spot of ALA
Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01034
3D densitogram of 80,160, 240, 320 and 400 ng/spot of ALA
Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01034
3D densitogram of 80,160, 240, 320 and 400 ng/spot of ALA
Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01034
Summary of method validation parameters
| Parameters | Result |
| Linearity range (ng/spot) n = 5 | 80–400 |
| Best fit values | |
| y-intercept | 0.00002600 |
| Slope | 0.00000825 |
| Goodness of fit | |
| Correlation coefficient(r 2) | 0.998 |
| Sensitivity | |
| LOD (ng/spot) | 18.022 |
| LOQ (ng/spot) | 54.612 |
| Precision | |
| Intra-day precision | 1.99 |
| Inter-day precision | 1.89 |
| % Recovery | 99.93 |
| Specificity | Specific |
LOD and LOQ
The values of detection and quantitation of ALA were found 18.022 ng/spot and 54.612 ng/spot respectively (Table 1).
Accuracy
Accuracy was calculated in terms of % recovery at each addition level with % RSD (Table 2) and the mean % recovery was estimated to be 99.93%.
Accuracy studies of ALA
| Amount of sample taken (ng/spot) | Amount of standard added (ng/spot) | Percentage of standard added | %Recovery | % Relative standard deviation |
| 90 | 80 | 80 | 101.38 | 1.09 |
| 90 | 100 | 100 | 99.68 | 0.77 |
| 90 | 120 | 120 | 98.74 | 1.81 |
Precision
Drug solution of 200 ng/spot concentration were analysed in triplicates for performing inter-day and intra-day precision. The consequence of the repeatability indicated no significant variation in intra-day (% RSD 1.99) and inter-day (% RSD 1.89) estimations. The values obtained were in the range (below 2%) (Table 1).
Specificity
To evaluate specificity of proposed method, drug content was determined in presence of their degradation products, more over there was no interference from excipients, present in commercial formulation, thereby confirming specificity of method (Fig. 6).
Analysis of marketed formulation
Densitogram of marketed capsules of ALA (INLIFE TM ) revealed only one spot at Rf 0.30 showing no interference from excipients of the capsule. The estimated amount of drug was found to 99.6% in capsules, which exhibited significant conformity with the label claim (300 mg), thereby re-emphasizing the fact that no interference of any excipients was there, indicating the method suitability for analysis of drug and its formulation.
Degradation studies
ALA exhibited varied degradation pattern under different stress conditions.
Acid degradation
In the study, six degradants were resolved at Rf 0.09, 0.15, 0.19, 0.27, 0.41, 0.73 along with drug peak at Rf of 0.33, indicating 20.90% degradation (Figs 5A, 7 and Table 3).
(A): Acid degradation of ALA: peak 1 (ALA Rf: 0.33), peak 2 (degradant Rf: 0.09) peak 3 (degradant Rf: 0.15), peak 4 (degradant Rf: 0.19), peak 5 (degradant Rf: 0.27), peak 6 (degradant Rf: 0.41), peak 7 (degradant Rf: 0.73); (B): Alkaline degradation of ALA: peak 1 (ALA Rf: 0.29), peak 8 (degradant Rf: 0.01), peak 9 (degradant Rf: 0.12), peak 3 (degradant Rf: 0.15), peak 10 (degradant Rf: 0.17); (C): Oxidative degradation of ALA: peak 1 (ALA Rf: 0.30), peak 11 (degradant Rf: 0.05), peak 9 (degradant Rf: 0.12), peak 3 (degradant Rf: 0.15), peak 12 (degradant Rf: 0.46), peak 7 (degradant Rf: 0.73); (D): Thermal degradation of ALA: peak 1 (ALA Rf: 0.31), peak 3 (degradant Rf: 0.15), peak 13 (degradant Rf: 0.84), peak 14 (degradant Rf: 0.94); (E): Photochemical degradation of ALA: peak 1 (ALA Rf: 0.30), peak 2 (degradant Rf: 0.09), peak 9 (degradant Rf: 0.12), peak 3 (degradant Rf: 0.15), peak 10 (degradant Rf: 0.17)
Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01034
(A): Acid degradation of ALA: peak 1 (ALA Rf: 0.33), peak 2 (degradant Rf: 0.09) peak 3 (degradant Rf: 0.15), peak 4 (degradant Rf: 0.19), peak 5 (degradant Rf: 0.27), peak 6 (degradant Rf: 0.41), peak 7 (degradant Rf: 0.73); (B): Alkaline degradation of ALA: peak 1 (ALA Rf: 0.29), peak 8 (degradant Rf: 0.01), peak 9 (degradant Rf: 0.12), peak 3 (degradant Rf: 0.15), peak 10 (degradant Rf: 0.17); (C): Oxidative degradation of ALA: peak 1 (ALA Rf: 0.30), peak 11 (degradant Rf: 0.05), peak 9 (degradant Rf: 0.12), peak 3 (degradant Rf: 0.15), peak 12 (degradant Rf: 0.46), peak 7 (degradant Rf: 0.73); (D): Thermal degradation of ALA: peak 1 (ALA Rf: 0.31), peak 3 (degradant Rf: 0.15), peak 13 (degradant Rf: 0.84), peak 14 (degradant Rf: 0.94); (E): Photochemical degradation of ALA: peak 1 (ALA Rf: 0.30), peak 2 (degradant Rf: 0.09), peak 9 (degradant Rf: 0.12), peak 3 (degradant Rf: 0.15), peak 10 (degradant Rf: 0.17)
Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01034
(A): Acid degradation of ALA: peak 1 (ALA Rf: 0.33), peak 2 (degradant Rf: 0.09) peak 3 (degradant Rf: 0.15), peak 4 (degradant Rf: 0.19), peak 5 (degradant Rf: 0.27), peak 6 (degradant Rf: 0.41), peak 7 (degradant Rf: 0.73); (B): Alkaline degradation of ALA: peak 1 (ALA Rf: 0.29), peak 8 (degradant Rf: 0.01), peak 9 (degradant Rf: 0.12), peak 3 (degradant Rf: 0.15), peak 10 (degradant Rf: 0.17); (C): Oxidative degradation of ALA: peak 1 (ALA Rf: 0.30), peak 11 (degradant Rf: 0.05), peak 9 (degradant Rf: 0.12), peak 3 (degradant Rf: 0.15), peak 12 (degradant Rf: 0.46), peak 7 (degradant Rf: 0.73); (D): Thermal degradation of ALA: peak 1 (ALA Rf: 0.31), peak 3 (degradant Rf: 0.15), peak 13 (degradant Rf: 0.84), peak 14 (degradant Rf: 0.94); (E): Photochemical degradation of ALA: peak 1 (ALA Rf: 0.30), peak 2 (degradant Rf: 0.09), peak 9 (degradant Rf: 0.12), peak 3 (degradant Rf: 0.15), peak 10 (degradant Rf: 0.17)
Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01034
(A): TLC plate showing specificity; (B): 3D densitogram of sample and standard drug showing no interference
Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01034
(A): TLC plate showing specificity; (B): 3D densitogram of sample and standard drug showing no interference
Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01034
(A): TLC plate showing specificity; (B): 3D densitogram of sample and standard drug showing no interference
Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01034
Number of degradants in different degradation media
Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01034
Number of degradants in different degradation media
Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01034
Number of degradants in different degradation media
Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01034
Stress degradation studies of ALA
| Degradation studies | Rf of ALA | Rf value of degradation products | % Degradation |
| Acidic-0.1N HCl, 45 min at 60°C | 0.33 | 0.09, 0.15, 0.19, 0.27, 0.41, 0.73 | 20.90 |
| Basic-0.1N NaOH, 45 min at 60°C | 0.29 | 0.01, 0.12, 0.15, 0.17 | 8.5 |
| Oxidative-6% v/v H2O2 for 45min (room temp) | 0.30 | 0.05, 0.12, 0.15, 0.46, 0.73 | 7.9 |
| Thermal-4h at 60°C | 0.31 | 0.15, 0.84, 0.94 | 19.42 |
| Photochemical (sunlight exposure) (48h) | 0.30 | 0.09, 0.12, 0.15, 0.17 | 5.38 |
Base degradation
In basic degradation, 8.50% degradation was observed within 45 min. Four degradation products were resolved with Rf values of 0.01, 0.12, 0.15, 0.17, from ALA peak (Figs 5B, 7 and Table 3).
Oxidative degradation
ALA was susceptible to oxidative degradation by 6% hydrogen peroxide over a period of 45 min resulting in 7.9% degradation. Five degradants were separated at Rf value of 0.05, 0.12, 0.15, 0.46 and 0.73 (Figs 5C, 7 and Table 3).
Conclusion
To estimate alpha-lipoic acid and its degradation products, a precise, accurate, selective HPTLC method of stability indicating, using derivatization, was developed. The method was validated as per latest ICH guidelines. The aimed method resolved 13 degradation products in stress degradation studies. The experimental study revealed unstable nature of alpha-lipoic acid in various degradation media. One degradation product (peak 3) with Rf of 0.15 was evident in nearly all degradation studies. Thus, the developed method was found to be accurate, responsive, specific, and offers many advantages in terms of reduced cost and run time. Stability data presented in the work may provide great help in the progression of formulations and have great commercial value for the industries regarding analysis of drug in bulk and capsule dosage form.
Author's contribution
All authors contributed substantially to the design of study, data analysis, interpretation, drafting plus revising the final manuscript for approval.
Funding
None.
Conflict of interest
Authors do not have any conflict of interest to declare.
Ethical approval
This study did not involve any animals or human subjects.
Acknowledgment
Authors are thankful to Maxtar Bio-Genics India, for providing sample of standard alpha-lipoic acid and to Mr. Dilip Charegaokar of Anchrom Laboratory, Mumbai for giving instrumental facilities for the work.
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