Chromatographic method for estimation of vitamin E from dried blood spot sample

We developed a selective bioanalytical RP-HPLC method for estimation of vitamin E from dried blood spot (DBS) sample, a potential technique which can be used for population-based epidemiological studies. Vitamin E was extracted from DBS by using liquid-liquid extraction technique with methanol (100% v/v) as reconstituting solvent for the residue. Alpha tocopheryl acetate was used as internal standard. Samples were analyzed directly on HPLC with C18 (250 × 4.6 mm × 5 μm) Phenomenex column. The mobile phase used was methanol to water (99:1% v/v) at a flow rate of 1.4 mL/min. The detector wavelength used was 292 nm. The retention time observed for vitamin E and internal standard was 10.225 ± 0.00075 min and 13.580 ± 0.00075 min respectively. The vitamin E calibration curve was found to be linear over the range of 0.625 to 60 μg/mL. The limit of quantification for vitamin E was found to be 0.1 μg/mL. Accuracy of the developed method was found to be 103.179%, 101.625%, and 100.174% with percentage of coefficient of variation of 0.0161, 0.0215, and 0.2790 for HQC, MQC, and LQC samples respectively which were within USFDA acceptance limit of ± 15 to ± 20%.The intraday and interday precision expressed as coefficient of variation were 0.0191–0.0841% and 0.0074–0.0252% respectively. The method represents a simple, rapid, specific, accurate, and precise method for estimation of vitamin E in human blood using DBS technique. The developed method can be further evaluated with respect to effect of matrix variability before it can be used in clinical setting.


Background
Vitamin E is an important lipid phase antioxidant. Oxidative stress in lipid component of cell leads to significant disturbance of cell homeostasis. Such stress has been identified as contributory factor in pathogenesis of various chronic diseases like, diabetes mellitus [1], Parkinson's disease [2], cardiovascular diseases [3], etc. Vitamin E helps to protect cell membrane against damage caused by free radicals and prevents the oxidation of low-density lipoprotein (LDL) cholesterol [4]. Vitamin E also plays a role in neurological functions and inhibition of platelet aggregation [5].
In spite of its widespread physiological importance and probable involvement in several chronic diseases, only few epidemiological studies are available for assessing role of vitamin E. A major hindrance in conducting such studies is the lack of availability of simple and cheap techniques for sample collection, preservation, and analysis.
Dried blood spot (DBS) refers to a blood sampling technique where little quantity of blood is spotted on a filter paper, dried, and preserved until analyzed [6]. This technique is well recognized in clinical laboratories for applications such as screening of inborn diseases like phenylketonuria in neonates. It has also been recently applied by many pharmaceutical companies in their drug development process, i.e., toxicokinetic and pharmacokinetic studies. The use of DBS in such studies is valuable as the sample volume required to be drawn every time is very less [7,8].
Literature review suggests an LC-MS/MS method is reported for estimation of vitamin E from dried blood spot; however, the use of LC-MS/MS technique itself limits the utility of the method pertaining to the availability of such facility [9][10][11].
Therefore, in present study, we aimed to develop and validate a method for estimation of vitamin E from DBS using high-performance liquid chromatography (HPLC) method that may enhance the utility and acceptability of the method.

Instrument and analytical conditions
The HPLC analysis was carried out using Shimadzu (LC-20AD) isocratic pump with UV detector (SPD-20A) run on the LC-Solution software. The column used was Phenomenex C 18 (250 × 4.6 mm, 5 μm), and detection was performed at 292 nm. Shimadzu (UV 1800) was used for UV visible analysis. Cooling microcentrifuge of REMI (RM 12 C) was used for centrifugation of samples.

Ethical approval
The need for ethical approval was waived as the study used left over blood samples from the laboratory.

Reagents and chemicals
Vitamin E and ascorbic acid were obtained from Sigma Aldrich (Mumbai, India). Triple distilled water was used for the HPLC experiment. HPLC grade methanol was purchased from SD Fine Chemicals Ltd. Other chemicals used were of AR grade and purchased from Sigma Aldrich (Mumbai, India). Whatman filter paper (grade 3, circles) 110 mm was obtained from GE Healthcare Life Sciences for blood spotting.

Preparation of standard stock solution
Accurately weighed 10 mg of vitamin E was transferred to 10-mL amber colored volumetric flask. Dissolved and diluted up to the mark with methanol (100% v/v) to give stock solution of 1000 μg/mL of vitamin E. From this solution, standard solutions with concentration of 60, 54, 45, 30, 9, 3, 1.25, and 0.625 μg/mL were prepared. The normal range of blood vitamin E level is 5.5 to 17 μg/ mL. The concentration range of 0.625 to 60 μg/mL was selected to cover this range. The stock solution of 100 μg/mL alpha tocopheryl acetate was prepared and added to each test tube of vitamin E standard solution to yield final concentration of 30 μg/mL of alpha tocopheryl acetate as internal standard.

Preparation of spiked blood samples and DBS sample
A range of blood samples with concentration of 60, 54, 45, 30, 9, 3, 1.25, and 0.625 μg/mL were prepared by adding  vitamin E stock solution of 1000 μg/mL. One hundred microliter of each blood sample was spotted on Whatman filter paper pretreated with 0.01% ascorbic acid. The same spot was spiked with 30 μL of 30 μg/mL internal standard solution. The cards were allowed to dry at room temperature and were stored at temperature below 4°C.

Elution from DBS sample
On the day of analysis, the blood spot was cut and placed in a 10-mL centrifuge tube. One milliliter of 2.5% sodium dodecyl sulfate (SDS), and 2 mL ethanol was added to each tube containing DBS cutout. The tube was vortexed for 20 min and then kept at room temperature for 10 min. Hexane (3 mL) was added to each tube and vortexed for 10 min. After this, each tube was centrifuged at 5000 rpm for 10 min to separate layers. The hexane layers were transferred to individual beakers, followed by evaporation of solvent under vacuum. The residue was reconstituted with methanol (100% v/v) and analyzed using HPLC method.

Chromatographic conditions
Chromatographic analysis was performed on a Phenomenex C 18 (250 × 4.6 mm, 5 μm) column. The mobile phase consisted of methanol to water (99:1% v/v), filtered through membrane filter, and degassed before pumping into HPLC system with following parameters. The flow rate was 1.4 mL/min. The detection wavelength was adjusted at 292 nm. Injection volume was 20 μL, and column was used at ambient temperature.

Method validation
The developed chromatographic method was validated for system suitability, specificity, sensitivity, accuracy, precision, recovery, and stability as per USFDA guidelines [12].

Method development and optimization
After trials of several mobile phase compositions, methanol to water (99:1% v/v) at a flow rate of 1.4 mL/min was

System suitability
For system suitability test, the mixture solution of 60 μg/ mL vitamin E and 30 μg/mL internal standard was prepared. Parameters such as theoretical plate count, tailing factors, resolution, and reproducibility in retention time (RT) for six repetitions of vitamin E and internal standard were also explored for compliance with the system suitability test. The result of system suitability analysis is shown in Table 1.

Linearity and range
Various concentrations from standard and its working solution of vitamin E and internal standard (ISTD) were prepared, and calibration graph was plotted between the peak area ratio and concentration (μg/mL). Three times of upper limit of vitamin E normal range (20 μg/mL) in body was taken as STD 1 (high quality control, HQC), i.e., 60 μg/mL, while 0.625 μg/mL (20 μg/mL divided by 32) is taken as concentration of LLOQ. A range of eight concentrations were prepared including HQC and LLOQ. The result of linearity and range is shown in Table 4. Overlay chromatogram of vitamin E with internal standard is shown in Fig. 5, and linearity graph of area ratio vs concentration is shown in Fig. 6.

Accuracy
Accuracy of the method was established using recovery technique with added internal standard. Five replicates of lower quality control (LQC), medium quality control (MQC), and HQC each were prepared and analyzed.  Table 5.

Precision
It is defined as the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of single homogenous volume of biological matrix. The precision of the developed method was studied by interday precision and intraday precision method. Five replicates of each concentration, i.e., LQC, MQC, and HQC were used for precision analysis. Intraday precision for high and medium QC samples was observed to be 0.0288 to 0.0191% which is within acceptance range of ± 15%, and for LQC sample, percentage of coefficient of variation (% CV) was found to be 0.0841% which is within acceptance range of ± 20%. Percentage CV for interday precision for HQC and MQC was found to be 0.01057-0.01923% and 0.01958-0.02484 % which is within acceptance range of ± 15%, and % CV for LQC sample was found to be 0.0501-0.1050% within the acceptance range of ± 20%. Percentage CV of intraday precision is shown in Table 6.

Recovery
It is defined as the detector response obtained from an amount of analyte added and to that extracted from the biological matrix. Repeatability was checked by injecting each concentration of six determinations such as LQC, MQC, and HQC respectively. Percentage recovery of vitamin E for LQC, MQC, and HQC was found to be 95.7713%, 91.1820%, and 93.0659%. The result of recovery is shown in Table 7.

Stability
Different stability studies for vitamin E, i.e., short-term stability, freeze thaw stability, long-term stability, and stock solution stability were performed as per USFDA guideline, and observed data are given in Table 8 which was observed within 15% for LQC and HQC acceptance criteria.
Developed method was validated as per USFDA guideline, and all parameters such as accuracy, precision, selectivity, recovery, and stability study were as per acceptance criteria.

Discussion
In our study, we used Whatman filter paper (grade 3, circles, 110-mm diameter) as a matrix for blood spotting. Several studies are available which have shown suitability of filter papers of specific grade for collection of blood samples in DBS technique [13,14]. The use of filter papers instead of specialized discs makes the method more approachable for many researchers, where supply of specialized discs may be limited.
As per USFDA guideline for validation of bioanalytical method, the method is specific as no interference was found for vitamin E or ISTD with the biological matrix and percentage CV for vitamin E (LLOQ), and internal standard was found to be within the acceptance range. The method was found to be linear over the concentration range of 0.625 to 60 μg/mLwith R 2 value of 0.9979. Intraday accuracy and interday accuracy for vitamin E is within the acceptance range of % CV ± 15% and ± 20% for MQC, HQC, and LQC respectively; hence, developed method is accurate. Method is reproducible, as intraday precision and interday precision for high and medium QC samples is within the acceptance range of ± 15%, and for LQC sample, % CV was found to be within acceptance range of ± 20%. Good recovery was found within the range of 91.1820 to 95.7713% with % CV 0.2074 to 0.3971 for all QC samples. The stability study data for vitamin E, i.e., short-term stability, freeze thaw stability, long-term stability, and stock solution stability were observed within acceptance criteria of 15% for LQC and HQC.
The existing method for analysis of vitamin E by DBS involves use of LC-MS technique. Sensitivity of LC-MS method is higher as compared to that of our newly developed method. However, the cost of analysis and availability of LC-MS instrument are a major limitation of the technique. The developed method is cost effective, and HPLC instrument is widely available.

Conclusion
Method was developed in mobile phase methanol to water (99:1% v/v), flow rate 1.4 mL/min at 292 nm with alpha tocopheryl acetate as an internal standard. Developed method was validated as per USFDA guideline.
The developed method was found to be highly accurate, specific, and precise. Stability studies like short term, long term, freeze thaw, and stock solution were also performed, and analyte was found to be stable. Thus, it can be concluded that vitamin E can be estimated from dried blood spot by RP-HPLC method.

Implication for health practice and research
Vitamin E being important lipid phase antioxidant mechanism, its derangement may be one of the factors in many recent disease breakthroughs. However, due to unavailability of suitable method, population-based epidemiological studies are missing. The method developed by us can be a valuable tool in field-based epidemiological studies, where immediate processing of the sample is not possible. The blood spot collection is a subject friendly, compact, and easy to store method as compared to collection of wet samples. Moreover, RP-HPLC analysis makes it economic and widely adaptable.