Quantitative Determination of Underivatized a-Tocopherol in Cow Milk, Vitamin and Multivitamin Drugs by GC-FID 2009, 70, 665–670
Yucel Kadioglu1,&, Fatma Demirkaya1, A. Kursat Demirkaya2 1 2
Department of Analytical Chemistry, Faculty of Pharmacy, Ataturk University, 25240 Erzurum, Turkey; E-Mail:
[email protected] Department of Food Hygiene and Technology, Faculty of Veterinary, Atatu¨rk University, 25240 Erzurum, Turkey
Received: 10 September 2008 / Revised: 22 April 2009 / Accepted: 23 April 2009 Online publication: 20 June 2009
Abstract A simple and rapid method for direct determination of a-tocopherol (a-T, vitamin E) in pharmaceutical preparations (vitamin and multivitamin tablets) and cow milk obtained from different villages of Erzurum in Turkey was developed and validated by GC-FID. Separation of underivatized a-T in pure substance, milk samples, vitamin and multivitamin tablets was performed in about 8.4 min, using an HP-5 capillary column. The range of quantification for the GC-FID was 1–30 lg mL-1. Within-day and between-day precision (RSD %) were less than 8.5%, and accuracy (relative error) was less than 11.0% (n = 6). LOQ and LOD values were found to be 0.35 and 0.30 lg mL-1, respectively. The developed method was applied directly and easily to the analysis of a-T in vitamin and multivitamin preparations and cow milk. RSD values were found to be 6.59% (Grandpherol soft gelatine capsule: 200 I.U.), 0.59% (Megadyn film tablet: 10 mg) and 1.54% (Supradyn drage: 10 I.U.). The developed method was also applied to cow milk samples and mean values of a-T content was found 2.99 lg mL-1 in cow milk samples. This developed and validated GC–FID method, in conjunction with other methods, could be successfully applied for routine laboratory because of its simplicity, rapidity, sensitivity, precision and accuracy.
Keywords Gas chromatography-flame ionization detection Vitamin preparations Cow milk a-Tocopherol
Introduction Vitamin E is a term used to designate a family of related compounds (tocopherol Limited Short Communication DOI: 10.1365/s10337-009-1184-y 0009-5893/09/08
and tocotrienols). It is a common structure with a chromanol head and phytyl tail (a-, b-, c- and d-tocopherol and also a-, b-, c- and d-tocotrienols) [1, 2].
a-tocopherol (5,7,8-trimethyltocol) is the most biologically active form of vitamin E. It functions as a chain-breaking antioxidant that prevents the propagation of free radical reactions [3, 4] and it is one of the most important fat-soluble antioxidants in biological systems [1, 5]. Vitamin E is found in milk in four main forms, a, b, c and d-tocopherol (a, b, c and d-T) but the major form of vitamin E in milk fat is a-T. The tocopherols have antioxidative properties with an important role in oxidative stability of milk. Content of a-T in cow milk is of significant relevance with the quantitative of a-T taken with cow food [6–8]. Many diseases, such as atherosclerosis, stroke, heart disease, cancer, rheumatoid arthritis, Alzheimer’s disease, Parkinson’s disease, diabetes mellitus types I and II, and even obesity have been proposed to be a result of an excess formation of free radicals [9–16]. Vitamin E, which is capable of detoxifying free radical species, may be helpful in the prevention and/or treatment of these conditions. Numerous epidemiological studies have demonstrated an association between higher intakes or higher blood concentration of vitamins and a lower incidence of certain degenerative diseases [16]. For this reason, there is an enormous public interest in vitamin supplements. Also an industrial pressure with limited control over marketing and
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quality exist. Vitamin E is taken with food. But if this intake is insufficient or if special dietary requirements exist, multivitamins or vitamin preparations can be taken in order to prevent vitamin deficiency. Such numerous preparations are often formulated available on the market. To clarify the roles of vitamin E, one of the fat-soluble antioxidant vitamins, it is essential to have simple and rapid analytical methods available for the measurement of a-T in a routine manner of drugs and food control [17]. Development of analytical methods which work in more rapid, simple and reliable manner than the existing ones are one of the objectives of analytical chemistry. Several methods have been reported for the determination of a-T including voltametric [18], spectrophotometric [19], chromatographic [20–23] methods in pharmaceutical preparations. In milk and dairy products, LC with different detectors is the most widely used technique to determine a-T [6, 17, 24–30] and also flourescence [31], spectrophotometric [32] and LC-MS [33] methods have been reported. An extensive survey of the literature showed that no direct GC-FID method for the analysis of a-tocopherol without derivatization in pharmaceutical preparations and milk samples has been reported. GC has been used at a lesser extent because the GC analysis of tocopherols normally implies formation of the trimethylsilyl derivatives prior to the chromatographic injection. Recently, derivatizations have been done by heating a purified extract with N-methyl-N-trimethyl-silyltrifluoroacetamide (MSTFA) [34–36], N,O-(bis-trimethylsilyl) trifluoro acetamide (BTSFA)-trimethyl-chlorosilane (TMCS) (10:1 v/v) [37] and with trisil Z-reagent (trimethylsilylimidazole-pyridine) [38]. We believe that the availability of a GC-FID system for the determination and quantification of a-T without derivatization with its increased simplicity will be very useful for the determination of a-T in pharmaceutical preparations and cow milk samples. For determination of underivatized a-T, this study describes a developed and completely validated GC-FID method. The validation of the method was carried out by establishing specificity, linearity,
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recovery values, limit of detection (LOD), limit of quantification (LOQ), within-day and between-day precision and accuracy according to International Conference on Harmonization guidelines for validation of analytical procedures [39]. We indicated the possibility of determination of a-T without derivatization and provided GC elution and resolution with considerable accuracy and sensitivity of analyte easily applied to commercial tablets and cow milk samples containing a-T.
Experimental Reagents and Chemicals a-Tocopherol (a-T) reference substance (95% purity) was purchased from Sigma (St, Louis, MO, USA) and LC grade ethanol (99.8%) and all other analytical grade chemicals were purchased from Merck (Darmstadt, Germany). Drugs containing a-T were obtained from the local market; Grandpherol soft gelatine capsule, containing about 200 I.U. (134.23 mg), Megadyn film tablet, containing about 10 mg and Supradyn drage, containing about 10 I.U. (6.71 mg). All gases were supplied by Havas (Ankara, Turkey).
Equipment The GC-FID method was performed with an Agilent 6890N Network GC equipped with a flame ionization detector (FID), an Agilent 7683 series autosampler and Agilent Chemstation software. The chromatographic separation was achieved using an HP-5 capillary column with 0.25 lm film thickness (packed (5%-Phenyl)-methylpolysiloxane 30 m, 0.32 mm 9 25 lm, USA). The splittless injection mode was used with nitrogen carrier gas and the flow rate of the carrier gas was kept constant during run at 2 mL min-1. The injector volume was 2 lL. The hydrogen (40 mL min-1) and synthetic air (400 mL min-1) were used as auxiliary gases for the flame ionization detector. The injector and detector temperatures were set at 300 °C. The oven tempera-
ture programs: initial temperature 120 °C, hold 1 min at this initial temperature, then ramped 28 °C min-1 to final temperature 350 °C and held at this final temperature for 3 min.
Preparations of Stock and Standard Solutions a-T standard solutions were prepared daily from stock solution (400 lg mL-1) up to final concentrations of: 1, 3, 5, 7, 10, 20 and 30 lg mL-1 in ethanol. The solutions were stored at 4 °C in dark bottles until analysis. The quality control samples (QC) were prepared at 2, 15 and 25 lg mL-1 concentrations. These solutions were used as quality control for the purpose of checking reproducibility of analyte in the daily analyses of standard samples.
Procedure for Pharmaceutical Preparations Samples of pharmaceutical preparations containing Megadyn film tablets, Supradyn drage and Grandpherol soft gelatin capsule containing a-T separately were prepared. Drugs solutions of Megadyn film tablets and Supradyn drage were prepared in ethanol, respectively, by accurately weighing and dissolving. Drug solution of Grandpherol soft gelatine capsule was prepared in ethanol by accurately transferring the content of the capsule. These solutions were sonicated and filtered through a Phenomenex filter (0.45 lm 9 25 mm) and stored in dark glass flasks, in order to protect them from light, and kept under refrigeration. Approximate dilutions were made (final concentration of drugs sample was 10 lg mL-1) and analyzed by GC-FID.
Procedure for Cow Milk Samples Samples of cow milk was transferred into a dark glass tube (to protect the vitamin against oxidation) and ethanol was added and the mixture was sonicated for 5 min to disrupt the proteins
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and phospholipids and then a-T from this mixture was extracted three times with hexane and dichloromethane mixture (9:1). The three organic phases were combined and transferred into another glass tube and evaporated to dryness at room temperature under nitrogen. The residue was dissolved in 1 mL ethanol and analyzed by GC-FID.
Statistical Analysis The SPSS 11.5 Version package was used for the statistical analysis.
Results Method Development The method development for the assay of a-T was based on its chemical properties. a-T (5,7,8-trimethyltocol) is an apolar molecule, therefore, ethanol was used as diluent. The capillary column coated with 5% phenyl, 95% dimethylpolysiloxane is a good choice for separation of this analyte since they elute as symmetrical peaks at a wide range of concentrations. The GC–FID parameters used in the method development were based on the boiling point. The injection port and detector temperature were set at 300 °C. Different temperature programs were investigated for exception of matrix interference. At the end of this investigation, the best temperature program was selected for a good resolution and thus all experiments were carried out at the oven temperature program described above. The good resolution of the peaks (analyte peak and interfering peaks in pharmaceutical preparations and cow milk) was not obtained when the ramp rate was less or more then 28 °C min-1. The head pressure was set to ensure a hydrogen flow of 40 mL min-1. The splitness mode was chosen. The solvent, column and acquisition parameters were chosen to be a starting point for the method development. a-T was directly analyzed without derivatization using these parameters and the separation with GC was perfectly achieved. The retention time of a-T was approximately Limited Short Communication
Fig. 1. Typical GC-chromatograms of three milk samples from cow in farms and three pharmaceutical preparation: a Grandpherol soft capsule solution (10 lg mL-1a-T). b Supradyn drage solution (10 lg mL-1a-T). c Megadyn film tablet solution (10 lg mL-1a-T). d Farm 11. e Farm 3. f Farm 12
8.4 min with good peak shape. The peak of excipients in chromatograms of pharmaceutical preparations (tablet, drage and gelatine capsule) and milk was not observed on the same retention time as a-T. That is, a-T was resolved from other interfering peaks. No further optimization of the method was required. Additionally, the concentration of analyte chosen in the peak response during the development of the method was shown to be significant with preliminary precision and linearity studies. Typical chromatograms obtained from solutions of the pharmaceutical preparations (tablet, drage and gelatine capsule) and milk samples containing a-T are shown in Fig. 1.
Linearity/Range of Quantification Linearity of the assay was demonstrated over a concentration range of 1–30 lg mL-1 a-T in six replicates at seven concentrations (1, 3, 5, 7, 10, 20 and 30 lg mL-1). The linearity was evaluated by linear regression analysis, which was calculated by least square regression method. The linear regression equation (with standard error of intercept (Sa: 0.0331) and slope (Sb: 0.0491)) was y = 1.0797 x - 0.0495 (y: the peak area, x: the concentration of a-T). The correlation coefficient was r = 0.9998. The limit of detection (LOD) defined as signal/noise = 3 in the method was
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Table 1. Determination of a-T in real samples Real samples
n
Found a-T content ± SD
Commercial preparations Grandpherol soft capsule 134.23 mg 10 132.6* ± 0.79 (200 I.U.) Megadyn film tablet 10 mg 10 9.97* ± 0.66 Supradyn drage 6.71 mg (10 I.U.) 10 6.76* ± 1.04 Milk samples collected from different cow farm. Milk 13 29.9** ± 19.2
Fat content (%)
Recovery (%)
–
98.77
– –
99.70 100.7
3.24 ± 0.97
–
n number of determination, *mg, **lg 10 mL-1, SD standard deviation of determinations
found to be 0.30 lg mL-1. The limit of quantification (LOQ) defined as signal/ noise = 8 in method was found to be 0.35 lg mL-1. Both accuracy and precision of these values were good within the proposed criteria (RSD % < 20%).
Precision (Repeatability and Intermediation Precision) The precision of the analytic method was determined by repeatability (within-day) and intermediate precision (betweenday). The precision study was examined by analyzing three different samples (2, 15 and 25 lg mL-1) by only one operator. The repeatability was evaluated by analyzing six times within 1 day, whereas intermediation precision was evaluated by analyzing once daily for 6 days. The RSD values for within-day precision were
