A Laboratory Experience in Analysis of Seized Medicines in the Republic of Macedonia

A counterfeit medicine is defined as a medicinal product which is manufactured by an illegal manufacturer or deliberately mislabeled with respect to identity of registered product. Once they enter the market, those medicines could pose a serious public health risk in a way that they do not deliver the desired effect and/or their use could lead to unexpected adverse effects, such as: anaphylaxis or developing resistance to the medicinal product. Therefore, fighting the entrance of counterfeit medicines in the country presents a significant national issue and requires a well-organized health system as well as market surveillance regulation. The Department for Medicines Quality Control at the Institute for Public Health of the Republic of Macedonia was actively involved in combating counterfeit drugs. In the period from 2007 – 2013, fourteen samples seized from the Customs of Macedonia were submitted to the Bureau of Medicines (Ministry of Health) to be analyzed in the Department for Medicines Quality Control. The identification and determination of the content of active substances was successfully achieved using laboratory methods from the registration documentation provided by the manufacturers of the licensed finished medicinal products or the internal HPLC methods validated previously and intended for control of the potentially counterfeit products. The most of the seized medicines were in pharmaceutical form of tablet (85.8 %), labeled as “Viagra”, “Cialis” or “Levitra”, and only 7.1 % were undeclared. The 14.2 % of samples were false labeled for the active compound. Regarding to the composition of the seized samples, the results showed that the most frequently identified active substances were those for treatment of erectile dysfunction: sildenafil citrate (50.5 %), tadalafil (21.4 %) and vardenafil hydrochloride trihydrate (7.2 %). The most of the analyzed products contained the active substance (64.3 %) outside the acceptable 95 % to 105 % margin of deviation from the declared value. The assay results for sildenafil citrate in the seized tablets were in range 52.1 % - 70.6 % from the declared content. These deviations verified the suspicion of counterfeit. However, for definite confirmation, more discriminating analytical techniques are needed, such as the near infrared spectroscopy (NIR) and/or mass spectrometry (MS). Additionally, the limited resource of reference standards in national quality control laboratories requires more extensive collaboration with international organizations

Evaluation of chromatographic conditions for simultaneously determination of Emtricitabine and Tenofovir

A fixed-dose combination of two antiretroviral drugs (tenofovir and emtricitabine) used for the treatment of HIV is already present on the European market. However, monographs of emtricitabine and tenofovir are not included in the actual European Pharmacopoeia. There is no official method for quality control of pharmaceutical dosage forms containing both emtricitabine and tenofovir. Therefore, the aim of this study was to optimize and to propose chromatography conditions for simultaneous identification and determination of emtricitabine and tenofovir active compounds in pharmaceutical dosage forms suitable for routine analysis in the quality control laboratories. In this study we used seven different ODS columns and we evaluate their efficiency in emtricitabine and tenofovir analysis. Testing for identification, specificity, selectivity, resolution and suitability was according to requirements in the ICH Q2(R1) Guideline (ICH, 2019). The emtricitabine and tenofovir can be successively separated with RP–HPLC octadecylsylil columns. Our experimental results showed that the optimal chromatographic conditions for simultaneous HPLC analysis of emtricitabine and tenofovir are achieved with mobile phase composed of 30% acetonitrile and 70%, water acidified with o-phosphoric acid at pH 2.6, at flowrate 1 mL/min, column oven set at 30ºC, with specific UV detection at 260 nm for tenofovir and at 284 nm for emtricitabine. Optimal results were obtained with Lichrospher 100 C18 (250 mm x 4 mm) and Purospher C18 end capped (150 mm x 4.6 mm) columns

A validated isocratic RP-HPLC method for determination of linezolid in pharmaceutical dosage forms

Linezolid is an oral and parenteral antibiotic that belongs to a new group of synthetic antibiotics known as fluorinated oxazolidinones. Linezolid has been assayed in dosage forms by spectrophotometry, liquid chromatography, high–performance thin–layer chromatography and micellar electrokinetic chromatography (Mohapatra et al., 2011). However, there is yet no monograph on linezolid in the current European Pharmacopoeia. Therefore, we aimed to develop simple, fast and reliable RP-HPLC method for determination of linezolid in dosage forms in the presence of its degradation products. The method performance was further fully validated according to requirements in the ICH Q2(R1) Guideline (ICH, 2019). Chromatographic separation was performed on a reversed-phase column Agilent ZORBAX SB C18 (250 x 4.6 mm I.D., particle size 5 μm), in an isocratic mode. The mobile phase consisted of a mixture of methanol and water acidified with o-phosphoric acid, pH 2.6, 50:50 (V/V). The flow rate was kept at 1.0 mL/min. Wavelength was selected by scanning a standard solution of linezolid over 200–400 nm using Model Lambda 12 (Perkin Elmer) UV-visible spectrophotometer and the wavelength of 254 nm was chosen for detection of linezolid. The injection volume was 20 μL. All separations were performed at a temperature of 30°C ± 2°C. The proposed RP–HPLC method allows simple, accurate and precise determination of linezolid in pharmaceutical dosage forms, in the presence of its degradation products and related compounds. The advantages of the method include short run time, simple sample and mobile phase preparation, isocratic mode of elution, and excellent peak symmetry. Therefore, the developed method can be applied for the routine analysis for determination of linezolid in pharmaceutical dosage forms in quality control laboratories

A Simple Method for Determination of Protodioscin in Tribulus Terrestris L. and Pharmaceuticals by High-Performance Liquid Chromatography Using Diode-Array Detection

The Tribulus terrestris L. (Zygophyllaceae) is a known medical plant used in traditional and folk medicine worldwide. The steroid saponine protodioscin, an active component found in this plant, serves as a marker for quality control of plant raw materials. In this study, we developed a simple and selective method for determination of protodioscin using convenient High-Performance Liquid Chromatographic (HPLC) laboratory equipment. The detection was performed by Diode-Array Detector (DAD). The proposed method was fully validated and according to the validation results, it was accurate and precise. It was proven to be linear over a protodioscin concentration range of 10,9 to 544.9 μg/mL. The low values of limit of detection (LOD) and limit of quantitation (LOQ) demonstrated adequate sensitivity (16.0 μg and 48.6 μg protodioscin per g plant material, respectively). The proposed method was successfully applied for quality control of a raw plant material intended for use in pharmaceutical industry, as well as for determination of protodioscin in a commercially available pharmaceutical formulation. The positive identification of protodioscin in analyzed samples was done by comparison of retention times of chromatographic peaks and their UV spectra. The content of protodioscin in analyzed samples was: 0.65 – 0.73 % in a raw plant material, and 0.38 % in tablets, respectively

A Simple Isocratic RP-HPLC Method for Determination of Nitisinone in Pharmaceuticals

Nitisinone is a reversible inhibitor of 4-hydroxyphenylpyruvate dioxygenase and an active substance in the orphan drug used for the treatment of hereditary tyrosinemia type I, a rare genetic disease caused by mutations in the gene for the enzyme fumarylacetoacetase. The aim of our study was to develop a simple and accurate RP–HPLC method with UV detection for routine determination of nitisinone in commercially available pharmaceutical forms. The chromatographic separation was achieved on a reversed-phase column Purospher STAR® RP–8 end–capped (150 x 4.6 mm i.d., particle size 5 μm), with a mobile phase consisted of a mixture of acetonitrile and water acidified with o-phosphoric acid with pH adjusted to 3.0, 65:35 (V/V), filtered through 0.45μm nylon filter. The flow rate was kept at 1 mL/min. A diode array detector measured the UV absorbance at 272 nm. The injection volume was 10 μl. The method was validated by a determination of system suitability, specificity, linearity, range, accuracy, precision, detection limit and quantitation limit. Then, the method was applied for determination of nitisinon in commercially available capsules. The proposed RP-HPLC method allows a simple, accurate, precise and rapid determination of nitisinone in pharmaceuticals. The advantages of the method include simple sample treatment, good precision (RSD less than 2%) and high recovery (greater than 99%). The method could be recommended for routine analysis in quality control laboratories, in stability studies as well as for the evaluation of potentially counterfeit capsules containing nitisinone

Validated HPLC Method for Determination of Sildenafil in Pharmaceutical Dosage Forms

Sildenafil is oral drug used primarily to treat male sexual function problems (impotence or erectile dysfunction) since becoming available in 1998. It is a potent and selective inhibitor of cGMP specific phosphodiesterase type 5 (PDE5) in the corpus cavernosum, where PDE5 is responsible for degradation of cGMP. Sildenafil has a peripheral site of action on erections. This substance has no direct relaxant effect on isolated human corpus cavernosum but potently enhances the relaxant effect of NO on this tissue. However, there is no analytical method for determination of this active compound in pharmaceutical preparations in the current European and US Pharmacopoeia. The aim of this study was to develop and validate HPLC method for sildenafil analysis in pharmaceutical dosage forms. HPLC analysis was performed using a Schimadzu LC-2010 chromatographic system (Schimadzu, Kyoto, Japan) consisting of a LC-20AT Prominence liquid chromatography pump with DGU-20A5 Prominence degasser, a SPD-M20A Prominence Diode Array Detector, RF 10AXI fluorescence detector and a SIL-20 AC Prominence auto sampler. Data analyses were done using Class VP 7.3 Software. The elution was carried out on a column Hypersil BDS-C18 (125 x 4 mm i.d., 5 mm), mobile phase consisted of phosphate buffer (20 mM, pH 2.8)-acetonitrile (71:29, V/V), flow rate 1.5 mL min-1, at controlled temperature (25oC) and auto sampler temperature at 4oC. Detection of sildenafil was carried out at 285 nm. Commercially available, film-coated tablets, containing 50 mg sildenafil as sildenafil citrate, were used in this study. The method was fully validated according to the ICH (International Conference on Harmonization) guidelines by determination of linearity, precision, accuracy, limit of detection and limit of quantification. Linearity of the method was tested in the range of: 2 – 100 mg mL-1 sildenafil. Experimental data showed high level of linearity which was proved with the value for the correlation coefficient (R2 = 0.9994). Limit of detection (LOD) and quantification (LOQ) of the method were tested in the range of: 20 – 200 ng mL-1 sildenafil. The results were: 0.23 ng and 0.68 ng for LOD and LOQ, respectively (9.2 ng mL-1 and 27.2 ng mL-1 for LOD and LOQ, respectively, obtained with 25 mL injected). Selectivity of the method was proved with the chromatographic peak resolution obtained between sildenafil and tadalafil (Rs = 10,5) Mean recovery for sildenafil was between 99,74% and 100,88% indicating that the developed method was accurate for determination of sildenafil in pharmaceutical formulation. The proposed method was successfully applied for determination of sildenafil in film-coated tablets, containing 50 mg sildenafil as sildenafil citrate. The results of the validation demonstrated that the proposed analytical procedure is accurate, precise and reproducible for sildenafil analysis in pharmaceutical dosage forms. Furthermore, this procedure is relatively inexpensive and simple and is particularly suitable for routine analyses when tandem mass spectrometric detection is not available. Additionally, it is important to mention that decreased consumption of organic solvent considerably reduces the laboratory expenses

Determination of cannabidiol and Δ9tetrahydrocannabinol in Cannabis sativa L. preparations present

The aim of this study was to apply in-house HPLC/DAD method on the Cannabis sativa L. preparations present in the European market and to control their quality. The satisfactory chromatographic data proved that our in-house gradient HPLC/DAD method can be successfully used for determination of the active substance CBD and traces of THC in Cannabis sativa L. preparations. The results obtained for the analyzed samples of Cannabis sativa L. preparations showed higher amounts of CBD than declared value in the certificate (differences ranged from 9.7% m/m to 25.4% m/m). In two samples, the amount of THC found was under allowed maximum limit of 0.20%, while in three samples the amount was above that value. Not all the samples tested have composition as declared in the certificate, although they all origin from a European manufacturer and are present in the European market

Процентуална застапеност на капсаициноиди во плодови од Capsicum sp. култивирани во Република Македонија

Капсаицинот е главен претставник од групата на протоалкалоиди, капсаициноиди. Формирањето на капсаициноидите е специфична особина карактеристична само за секундарниот метаболизам на растенијата од родот Capsicum. Лутите пиперки се карактеризираат со присуството на ванилил конјугати од амиден тип, отсутни или заменети од страна на нивните нелути естри, изостери (капсиноиди) кај благите видови на пиперки. Пиперката Capsicum annuum L., fam.Solanace, по своето стопанско значење е една од водечките градинарски култури во Република Македонија. Таа се одгледува најмногу заради плодовите што се користат во исхраната преку целата година, во нивната ботаничка или технолошка зрелост. Поради тоа целта на овoј труд беше да се направи анализа на процентуалната застапеност на најзастапените капсаициноид и тоа капсаицин, дихидрокаспаицин и нордихидрокапсаицин во екстрактите добиени од плодовите на петнаесет различни генотипови на Capsicum annuum L., fam. Solanaceae

Determination of THC by HPLC/DAD in food supplement samples of hemp seed oil

The EU member states have different regulation in allowed limit of controlled compound delta-9-tetrahydrocannabinol (THC) in hemp seed oil, produced for consumption or as food supplement. The THC traces were analyzed in few samples by recommended HPLC isocratic method. Using different HPLC columns (C8 and C18) it was not succeeded to separate THC from one component present also in the other plant oils (flax seed oil and rape seed oil), used as blank samples. This unidentified compound has not quite characteristic UV spectrum and it is similar with the UV spectrum of THC. Then we developed the gradient mode HPLC method and succeeded to separate THC’s peak (18.6 min) from this compound’s peak (19.4 min) with suitable resolution (Rs is 2.36). The obtained results for found quantity of THC in tested samples of hemp seed oil are in range: 2.66 mg/L – 9.84 mg/L. These results are in correlation with the already published data for this kind of samples

Determination of Ferulic Acid with RP-HPLC Method in Oral Solution Containing Extract of Ferula Assafoetida L. and Viscum Album L.

Ferula assafoetida L. is a medicinal plant widely used in the traditional medicine. Roots of Asafoetida, produces natural antiviral drug compounds that kill the swine flu virus, H1N1. The aim of this study was to develop and validate a RP-HPLC method for quality control of commercially available oral solution containing extract of Ferula assafoetida L. and Viscum album L. Determination of the content of Ferula assafoetida L. extract in the oral solution was made by determination of ferulic acid, one of components identified in Ferula assafoetida L. plant. The chromatographic separation was achieved on a reversed-phase column Purospher STAR RP -18e (150 x 4.6 mm i.d., particle size 5μm), gradient run (using acetonitrile and 0.01 mol L-1 orthophosphoric acid as a mobile phase), at 25°C temperature. The flow rate was kept at 1.5 ml min-1. Detection of ferulic acid was carried out at 316 nm using photodiode array detector. The proposed method was fully validated according to the ICH guidelines in terms of accuracy, precision, linearity and range. The obtained data for precision, (RSD of 9.69%), and accuracy (recovery of 104.39%) are suitable for this kind of analysis. The linearity of the proposed method was tested in the range of 0.1 – 1.8 μg mL-1 with regression coefficient of 0.9995 obtained. These validation results demonstrate the suitability of the method for quality control of this oral solution with expected content of ferulic acid of about 0.0001%, m/m (1 ppm). This developed HPLC method was proven to be precise, specific, sensitive, and accurate for routine quality assessment of raw material plant Ferula assafoetida L., its extract, and pharmaceutical products