See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/279201511
DETERMINATION OF UNDECLARED SILDENAFIL CITRATE AND TADALAFIL IN APHRODISIAC HERBAL PREPARATIONS BY TLC AND HPLC
Article · July 2015
READS 70
5 authors, including:
Magdi Awadalla Mohamed Shaza Wagiealla Shantier Faculty of Pharmacy, University of Khartoum University of Khartoum
34 PUBLICATIONS 37 CITATIONS 16 PUBLICATIONS 4 CITATIONS
SEE PROFILE SEE PROFILE
Elrasheed Gadkariem Esraa M.O. Ismail University of Khartoum University of Khartoum
22 PUBLICATIONS 5 CITATIONS 6 PUBLICATIONS 1 CITATION
SEE PROFILE SEE PROFILE
All in-text references underlined in blue are linked to publications on ResearchGate, Available from: Magdi Awadalla Mohamed letting you access and read them immediately. Retrieved on: 16 June 2016 RESEARCH ARTICLE Shantier et.al / IJIPSR / 3 (6), 2015, 688-696 Department of Pharmaceutical Chemistry ISSN (online) 2347-2154
International Journal of Innovative Pharmaceutical Sciences and Research www.ijipsr.com
DETERMINATION OF UNDECLARED SILDENAFIL CITRATE AND TADALAFIL IN APHRODISIAC HERBAL PREPARATIONS BY TLC AND HPLC
1,2 Safa Mohammed A. Saeed, 1Magdi Awadalla Mohamed, 1Shaza Wagiealla Shantier*, 1Elrasheed Ahmed Gadkariem, 1Esraa M.O. Ismail 1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Khartoum, SUDAN 2National Drug Quality Control Laboratory, Khartoum, SUDAN
Abstract
Liquid chromatographic methods, namely TLC and HPLC, were developed for the qualitative and quantitative analysis of undeclared Phosphodiesterase type 5 inhibitors in aphrodisiac herbal preparations. Thirty aphrodisiac herbal preparations, marketed in Khartoum City, were screened for
Sildenafil Citrate and Tadalafil. Nine of them were found to be adulterated with sildenafil citrate while tadalafil was detected in three preparations. HPLC analysis revealed that a single dose of the adulterated samples contain 165.46±0.79 mg of sildenafil or 60.76±0.60 mg of tadalafil. The use of such preparations could result in fatal side effects, knowing that the maximum daily dose for sildenafil and tadalafil is
100mg and 20mg, respectively.
Keywords: Herbal preparations; Adulteration; Sildenafil; Tadalfil; Liquid chromatography.
Corresponding Author Shaza Wagiealla Shantier Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Khartoum, SUDAN Email: [email protected] Phone: +249 912637509
Available online: www.ijipsr.com June Issue 688
RESEARCH ARTICLE Shantier et.al / IJIPSR / 3 (6), 2015, 688-696 Department of Pharmaceutical Chemistry ISSN (online) 2347-2154
INTRODUCTION Despite the huge development and mass production of chemically synthesized drugs, large sections of the population in developing countries still rely on traditional practitioners and herbal medicines for their primary care [1]. This is, in part, due to the perceived effectiveness and safety of herbal medicines. Nevertheless, deleterious effects associated with the consumption of herbal products are disclosed from time to time. The majority of these adverse effects were attributed to different reasons including adulteration with drugs [2, 3]. To date, a plenty of studies worldwide have reported the adulteration of aphrodisiac herbal preparations with PDE-5 inhibitors and their analogues [4-9]. We herein report unprecedentedly detection and determination of undeclared Sildenafil (Figure 1) and tadalafil (Figure 2) in some aphrodisiac herbal preparations marketed in Khartoum City, Sudan.
Fig. 1: Sildenafil chemical Structure Fig. 2: Tadalafil chemical Structure
MATERIALS AND METHODS Materials Thirty different herbal products, formulated as tablets, capsules, powder and honey samples, claimed to enhance sexual activity were collected from street, local practitioners (Ataras), honey centers, herbal centers and licensed companies in Khartoum city. Sildenafil working standard (as citrate) was kindly provided by General Medicines Co. (GMC), Sudan. Tadalafil working standard was kindly provided by Azal Pharmaceutical Industry, Sudan. The following chemicals were all obtained from Scharlau, Spain: Methanol Analytical grade; Methanol HPLC grade; Potassium dihydrogen phosphate; Chloroform; Ethyl acetate; N-Hexane; Acetonitrile HPLC grade; Acetone. Instrumentation HPLC Shimadzu – Japan consisted of Kromasil 100(4.6 mm × 250mm Column that contains 5 µm packaging L1), UV–Detector (SPD – 10AVP) adjusted at wavelength 290nm and Pump (LC- 10Ai). Available online: www.ijipsr.com June Issue 689
RESEARCH ARTICLE Shantier et.al / IJIPSR / 3 (6), 2015, 688-696 Department of Pharmaceutical Chemistry ISSN (online) 2347-2154
Thin-layer chromatography plates (Aluminum sheets 20x20cm pre-coated with silica gel 60F254); Merck- Germany. Chromatographic Conditions HPLC separation was carried out using the mobile phase composed of a mixture of potassium dihydrogen phosphate (0.05M) pH adjusted to 5.8, acetonitrile and methanol (30: 50: 20) (v / v ). The mobile phase was pumped through the column at a flow rate of 1 ml min-1. The solvent systems used in TLC were: N-Hexane: Ethyl acetate: Methanol (8: 6: 2 v/v), Methanol: Chloroform (1: 4 v/v) and Methanol: Ethylacetate (1: 2 v/v). Visualization was accomplished under UV light (254nm-360nm). Preparation of Standard Solution for TLC Analysis An approximate quantity of 0.4g of either sildenafil citrate or tadalafil working standards was dissolved in methanol or acetone and sonicated for three minutes (solution A). Preparation of Sample Solution for TLC Analysis An approximate quantity of 0.4g of each sample was suspended in methanol or acetone, sonicated, centrifuged and filtered through 0.45µm syringe filter disc (solution B). Sample Preparation for Quantitative Analysis Herbal Products Formulated As Powder and Honey An amount equivalent to 0.5g of each sample was suspended in methanol and the volume completed to 20 ml. The solution was sonicated for 30 minutes, centrifuged for 30 minutes at 3500 rpm and then filtered by using 0.45µm syringe filter disc (solution C). Herbal Products Formulated As Tablets and Capsules The average weight of five tablets or capsules was suspended in methanol and the volume was completed to 20 ml with methanol. The solution was sonicated for 30 minutes, centrifuged for 30 minutes at 3500 rpm and filtered using 0.45µm syringe filter disc. The filtrate was then diluted 5 to 10 v/v by methanol (solution D). Procedures Qualitative Analysis of Prepared Sample Solution Using Developed TLC Method The three mobile phases were prepared and transferred into different chromatographic tanks; each tank was tightly closed and allowed to equilibrate for one hour. Each TLC plate was spotted with solutions A & B at a distance of 1cm from the bottom of the plate and allowed to dry .The plates were allowed to run upto 8cm height using the selected mobile phases, then dried and
Available online: www.ijipsr.com June Issue 690
RESEARCH ARTICLE Shantier et.al / IJIPSR / 3 (6), 2015, 688-696 Department of Pharmaceutical Chemistry ISSN (online) 2347-2154 examined under UV at λmax254 nm. The retardation factor (Rf) for each working standard was used as an identification parameter for any similar Rf values from the samples spots. Quantitation of Detected Sildenafil and Tadalafil in the Samples Solution Calibration Curve A mixture of sildenafil standard (0.02g) and tadalafil standard (0.02g) was dissolved in methanol and the volume was completed to 100ml. Serial dilutions were made using methanol to obtain 6 solutions of concentrations range (0.064 – 200 µg/ml for each standard). The HPLC procedure was carried out using the mobile phase composed of a mixture of 0.05M potassium dihydrogen phosphate (pH adjusted to 5.8): acetonitrile: methanol (30: 50: 20) (v / v / v). The resultant peak areas were plotted against the standard concentrations to check the linearity. In order to determine the content of sildenafil or tadalafil in the adulterated samples, solutions C and D were treated as under calibration curve. The resultant peak RT corresponding to either sildenafil or tadalafil were used for the confirmation of their presence as adulterants; on the other hand the peak areas were used for the quantitative estimation of the content of each adulterants depending on either direct sample/standard comparison or from the regression analysis data. Validation of the adopted HPLC method Precision The Within-day and between-days data were determined for three concentrations within the linearity range. RSD % values were calculated to assess method precision which should be < 2.
RESULTS AND DISCUSSION People, worldwide, hold a false perception about the safety of natural products and therefore prefer the use of herbal preparations rather than the marketed medicines. Several reports have disclosed the adulteration of herbal preparations with one or more of the medicines that are clinically used for the same therapeutic indication of the herbal preparation. In Sudan, the numbers of local practitioners and herbal companies have dramatically increased over the last decade and there have been an increasing demand of herbal products for sexual enhancement. This is greatly evidenced by the large number of different herbal preparations sold in the Sudanese market as alternatives to the PDE-5 inhibitors sildenafil and tadalafil. Thirty different aphrodisiac herbal products, marketed in Khartoum City were collected equally, from streets, local practitioners (Ataras), Honey centers, herbal centers and some companies specialized in herbal preparations. It was first planned to screen those thirty samples for the presence of
Available online: www.ijipsr.com June Issue 691
RESEARCH ARTICLE Shantier et.al / IJIPSR / 3 (6), 2015, 688-696 Department of Pharmaceutical Chemistry ISSN (online) 2347-2154 sildenafil citrate and / or tadalafil using TLC. Therefore, mixtures of solvents with different polarity and percentages were investigated. A mixture of N-Hexane: Ethyl acetate: Methanol (8: 6: 2 v/v/v) was first tried for analysis of the 30 samples. Three samples developed spots of a similar retardation factor to that of standard tadalafil (Rf = 0.63), nine samples developed spots of a smaller retardation factor (Rf = 0.16) and no spots were observed for the rest of the samples. Based on standard sildenafil retardation factor ((Rf = 0.16), the second set of above samples were proposed to be adulterated with sildenafil citrate. It was, thus, decided to try more polar system to bring the standard sildenafil citrate spot to a higher Rf. A mobile phase consisted of methanol: chloroform (1: 4 v/v) separated sildenafil citrate and tadalafil with Rf values 0.85 and 0.88 respectively. The third mobile phase consisted of methanol: ethylacetate (1: 2 v/v) showed the best separation for the developed spots with Rf values 0.43 and 0.83 for sildenafil citrate and tadalafil respectively. We hereby recommended this solvent system for routine TLC analysis of mixtures containing both sildenafil citrate and tadalafil. It is worth noting that TLC spiking test had further confirmed these preliminary findings. Having determined the samples that are adulterated with sildenafil citrate or tadalafil, we next directed our attention to further confirm the presence of these adulterants by HPLC method. To best of our knowledge, there are two reported HPLC methods for analysis of mixtures containing both sildenafil citrate and tadalafil [10, 11]. Out of the mobile phases cited in the reported methods, the mobile phase composition in [11] was found more promising to resolve both analytes from herbal matrix. Therefore, we decided to adopt the reported method [11] using the same mobile phase but changing the column length and introducing an extraction process with methanol. This adoption was expected to improve the resolution of the system through increasing the column efficiency (N), selectivity (α) and capacity factor [12]. Good resolution of both analytes from each other and from herbal matrix was obtained where sharp peaks at retention time 4.2 and 5.8 minutes were assigned to tadalafil and sildenafil citrate respectively (Figure 3).
Fig. 3: Typical HPLC chromatogram for Tadalafil and Sildenafil citrate standards mixture (40µg/ml) Available online: www.ijipsr.com June Issue 692
RESEARCH ARTICLE Shantier et.al / IJIPSR / 3 (6), 2015, 688-696 Department of Pharmaceutical Chemistry ISSN (online) 2347-2154
Successfully, the adopted HPLC method was proved to be suitable for separation of mixtures of sildenafil and tadalafil. The adopted HPLC method was then applied for the estimation of the content per dose of sildenafil or tadalafil in the adulterated samples. The adulterated samples showed intractable mixture of products eluted at retention times 2 – 3.5 minutes; peaks at 4.2 and 5.8 minutes which were assigned to the adulterant tadalafil and sildenafil citrate (Figure 4, 5).
Fig. 4: Typical HPLC chromatogram for sample adulterated with tadalafil
Fig. 5: Typical HPLC chromatogram for sample adulterated with sildenafil citrate
At this time, the validation of the adopted method was carried following the ICH guidelines [13]. The adopted method was found to be precise (RSD% < 2) and free from interference from other components of the herbal preparations. Furthermore, linearity test revealed that the adopted method is linear for sildenafil citrate (R2 = 0.9998) and tadalafil (R2 = 1.00) at a concentration range of 0.064 – 200 µg/ml. The system suitability tests were greatly evidenced upon analysis of the standard sildenafil citrate and tadalafil chromatograms parameters (Table 1). Table 1: Calculated system suitability parameters for the HPLC method (ICH guidelines)
Number of peak HETP Capacity chromatographic Parameter theoretical symmetry (mm) factor Resolution plates factor
Tadalafil 5555.389 22.222 0.93 4.0 5.73 Sildenafil 5761.946 23.05 1 5.5
Available online: www.ijipsr.com June Issue 693
RESEARCH ARTICLE Shantier et.al / IJIPSR / 3 (6), 2015, 688-696 Department of Pharmaceutical Chemistry ISSN (online) 2347-2154
Finally, the adopted method was applied in the quantification of the adulterated samples. Our findings revealed that, these samples contained 38.0 – 165.0 mg/dose and 39.0 – 60.7 mg/dose of sildenafil citrate and tadalafil, respectively (Table 2). Table 2: Summary of the content of tadalafil and sildenafil per dose in some adulterated samples Adulterant Sildenafil Tadalafil Sample Weight Dose / g Content % Content % code taken / g Content / dose Content / (w/w) ± (w/w) ± ± RSD% dose ± RSD% RSD% RSD% 1 5.4026 0.5 2.22 ± 0.05 0.1197±0.96 -- -- 2 9.1533 0.5 0.42 ±1.04 0.0384 ±2.94 -- -- 8 4.7059 0.5 -- -- 0.83±1.65 0.039 ± 4.65 13 20 0.5036 -- -- 0.29 ±0.02 0.058± 0.02 24 5 0.5185 1.36 ± 0.003 0.068 ±0.003 -- -- 25 0.7426 0.7426 22.3 ±0.417 0.165± 0.79 -- -- 28 13 0.5 -- -- 0.47 ±0.59 0.0607± 0.59
All the positive samples for tadalafil were found to contain over therapeutic dose (> 20 mg/dose) [14]. On the other hand, 56% of sildenafil-positive samples contained over therapeutic dose (> 100 mg/dose) [15]. It is worth noting that all preparations collected from licensed herbal companies (eight preparations) were found to be adulterated with sildenafil citrate or tadalafil. The serious side effects of sildenafil and tadalafil along with their potential drug-drug interactions and/ or drug-diet interactions render consumption of these adulterated herbal preparations very dangerous. It is worth noting that 67% of the adulterated samples are heavily advertised in the media. It, therefore, appeared that the channels delivering herbal preparations to the market are not strictly regulated by the authority body. An advantage of the adopted method is that it can be used for the routine analysis of sildenafil and tadalafil in pharmaceutical formulations using either drug as an internal standard for the other. CONCLUSION In this study, TLC and adopted HPLC method were used for identification and quantification of sildenafil citrate and tadalafil in 30 herbal preparations marketed in Khartoum City. Twelve preparations were adulterated with sildenafil citrate and tadalafil and 50% of these preparations were found to be overdosed, putting the health of patients at stake. The findings of the current studies clearly revealed that the manufacturers have clandestine intention to deceive and satisfy customers who are seeking for better sexual performance. These manufacturers also misguide public through objectionable advertisements via both print and electronic media. Thus, to protect
Available online: www.ijipsr.com June Issue 694
RESEARCH ARTICLE Shantier et.al / IJIPSR / 3 (6), 2015, 688-696 Department of Pharmaceutical Chemistry ISSN (online) 2347-2154 herbal consumers form unethical practicing, more effort should be elicited by the authorized persons to make the present regulations effective and to set new rules, if necessary. REFERENCES 1. World Health Organization (WHO). National Policy on Traditional Medicine and Regulation of Herbal Medicines. Geneva: Report of WHO global survey. 2005. 2. Chan K. Some Aspects of Toxic Contaminants in Herbal Medicines. Chemosphere. 2003; 52: 1361–1371. 3. Zhang J1, Wider B, Shang H, Li X, Ernst E. Quality of herbal medicines: challenges and solutions. Complement. 2012 Feb-Apr; 20(1-2):100-6. 4. Mu-Chang T, Jer-Huei L. Determination of Sildenafil Citrate Adulterated in a Dietary Supplement Capsule by LC/MS/MS. J. of Food Drug Anal. 2002; 10(2): 112-9. 5. Samuel R G, Cheryl L F, Karen A W. Analysis of undeclared synthetic phosphodiesterase-5 inhibitors in dietary supplements and herbal matrices by LC-ESI-MS and LC-UV. J. Pharm. Biomed. Anal. 2004; 36 (3): 525 - 33. 6. Oh SS, Zou P, Low MY, Koh HL. Detection of sildenafil analogues in herbal products for erectile dysfunction. J Toxicol Environ Health A. 2006; 69(21):1951-8. 7. Mei-Chih L, YI-Chu L, Yun-Lian L, Jer-Huei L. Identification of a Tadalafil Analogue Adulterated in a Dietary 125 Supplement. J Food Drug Anal. 2009; 17(6):451-8. 8. Pavan P, Shyam SA. Detection and estimation of synthetic PDE-5 inhibitors viz. Sildenafil and Tadalafil in marketed herbal aphrodisiacs New Trends in Pharm. Sci. 2014; 1:1-9. 9. Ji Hyun L, Hyung JK, Eunyoung N, Jung YK, Jung-Ah D, Chang-Yong Y et al. Identification and screening of a tadalafil analogue found in adulterated herbal products. J Pharm. Biomed. Anal. 2015; 103: 80-4. 10. Kannappan N, Deepth Y, Divya Y, Shashihanth MR. Method Development and Validation of Stability-indicating Methods for assay of Tadalafil and Sildenafil Citrate by HPLC. Int J ChemTech Res. 2010; 2(1): 329-333. 11. Amr MB, Abd El-Aziz B AE, Nesrine TL. Stability indicating methods for the determination of sildenafil in the presence of its degradation product. Int J Compr Pharma. 2011; 2 (7). 12. David H. Modern Analytical Chemistry. McGraw-Hill Companies. 2000 : 547-556.
Available online: www.ijipsr.com June Issue 695
RESEARCH ARTICLE Shantier et.al / IJIPSR / 3 (6), 2015, 688-696 Department of Pharmaceutical Chemistry ISSN (online) 2347-2154
13. International Conference on Harmonization (ICH). Harmonized Tripartite Guideline Validation Of Analytical Procedures :Text and methodology Q2(R1) Current Step 4 version Parent Guideline October 1994 page11,2. 14. Virginia PA, Andrew MP.Pharmacotherapeutics for Advanced Practice: A Practical Approach. 2006: 959. 15. MP. Pharmacotherapeutics for Advanced Practice: A Practical Approach. 2006: 959.Edward TB, Rick DK. Conn's Current Therapy, Expert Consult. 2013: Online page 967.
Available online: www.ijipsr.com June Issue 696