Analytical profiling of listed vitamin D brands in Australia
Report Draft
Analytical profiling of listed vitamin D brands in Australia
Supervisors:
Ayman AllahhamThilini ThrimawithanaJournal Name:
Total word count:
A total number of figures/tables:
Abstract (summary of the draft )
ObjectiveMethodsResultsConclusionKey wordsKeywords
Vitamin D3, HPLC, Friability, Disintegration, Dissolution
Introduction
Complementary medicines (CMs) are products that contain vitamins, minerals, herbs, nutritional supplements, natural products, homeopathic treatments, or aromatherapy preparations. People use CMs to manage specific health conditions, improve general health and well-being and enhance everyday performance (Popattia et al., 2021; Popattia et al., 2018). Australian people are among the highest countries using complementary medicines (CMs), either alone or alongside other conventional medicines (Cohen & Hunter, 2017). People use CMs either for general health improvement and treating self-limiting minor ailments or to manage some serious medical conditions. The use of complementary and alternative medicines has increased in the last few years in Australian society. It has been estimated that two out of three Australian adults are using CMs (Armstrong et al., 2011). A national survey reported that the use of CMs by Australian people in 2006 was 67% of the nations population (Popattia et al., 2018). Complementary medicines sales have increased more than triple in Australia, with over A$94 million sold of vitamin D supplements in 2010 (Bilinski & Talbot, 2014). Australian Bureau of Statistics showed that 5% of Australians used Vitamin D supplements in 2011-2012 (Australian Bureau of Statistics, 2013). The primary source of vitamin D is skin exposure to sunlight (Stalgis-Bilinski et al., 2011). However, sun exposure carries a risk of skin cancers (Cancer Australia, 2015) and the seasonal fluctuation, mainly in winter, reduces the chance of getting enough sunlight for vitamin D synthesis. Vitamin D fortification for diet is difficult (Jayaratne et al., 2013). It has been suggested that supplementation is the safe and effective way of maintaining normal vitamin D levels (SHRAPNEL & TRUSWELL, 2006). The Australian national health and medical research council recommends 400-600 IU of vitamin D per day to maintain a normal range of serum 25(OH) D (2020). Supplementation of 10002000 IU of vitamin D per day is recommended in Australia for those who have mild vitamin D insufficiency (Rejnmark et al., 2012).In Australia, vitamin D is marketed in several dosage forms, such as tablets or capsules. It is marketed as a single preparation or in combination with other medicines. Unless excepted or excluded, all therapeutic goods must be included by the Australian Register of Therapeutic Goods (ARTG). Based on the level of the risk of the therapeutic goods, TGA categorises the products included in the ARTG into two categories: registered and listed products. High-risk medicines must be registered, while low-risk products are listed only (Therapeutic Good Administration, 2022). Registered medicines are tested for quality, safety, and efficacy. On the other hand, Listed medicines are tested for quality and safety only. Most complementary medicines are listed under the ARTG rather than registered. TGA classifies vitamin D both as a low-risk and high-risk therapeutic good, depending on the strength. Low-dose vitamin D products are considered low-risk products. Consequently, they are Listed under the ARTG (Therapeutic Good Administration, 2017). The risk associated with therapeutic goods, including CMs, is managed by the processes of licensing manufacturers, assessing the products for safety before their release to the market, and post-market evaluation (Briggs, 2002).Registered medicines encounter comprehensive evaluation for quality, safety, and efficacy before releasing into the market. On the other hand, Listed CMs, including Listed vitamin D products, do not undergo evaluation prior to the release onto the market, face limited surveillance once released, and sponsors who break the regulations face no timely or effective consequences (Harvey, 2017). By these less stringent regulations the TGA relies upon, its expected that CMs sponsors do not necessarily meet the high standard obligations for quality and safety. In the data that was published by the TGA, the compliance activity audit by TGA has increased to more than double between the financial years (2014-2015) and (2015-2016). The compliance breaches of Listed CMs have increased from 73% to 80%, respectively (Harvey, 2017). According to TGO101, registered tablets or capsules must comply with uniformity of dosage unit and dissolution tests for active ingredients to demonstrate that the products are of good quality. However, Listed medicines are not required to comply with the uniformity of dosage unit test, and it can be replaced by a uniformity of weight (mass) test to fulfil the tests for uniformity, and dissolution test is required only for modified release goods (Therapeutics Goods Administration, 2020). This flexibility in the quality requirements for Listed medicines makes CMs sponsors choose the easy and less expensive tests to go with in order to comply with TGA standards. Vitamin D-containing supplements, manufactured under less strict quality standards, showed wide variations between the measured strength and what was claimed on the label (Wan et al., 2021).(763 words)HPLC methods and how it offers good analysis. Aim: Testing the quality of vitamin D brands.Experimental
Chemical and reagents
Acetonitrile, Ethanol ethanol (99%), and Methanol methanol were obtained purchased from --------, and Milli Q water was purified by reverse osmosis (RO) (Merck water purification system). Cholecalciferol (D3) reference standard purchased from ----------, Vitamin D3 tablets and capsules in different brands and batches were purchased from local pharmacies. All solvents were HPLC grade.
Apparatus
Analytical balance (----), Vortex mixer (----), Centrifuge (----), sonicator (---), High-performance liquid chromatography (1260 Infinity II), and Dissolution apparatus (-----) were used in sample preparation.
Standard solution preparation
The working standardA standard stock solution of vitamin D3 (100 mg/L) was prepared by dissolving 5 mg of the reference standard (Cholecalciferol (D3)) into a 50 ml volumetric flask and diluting it to the volume with methanol. From the standard stock solution, different standard solutions were prepared (0.5, 1, . 10 mg/L) were prepared using methanol as solvent. All prepared volumetric flasks used were covered with aluminium foil to protect drug from degradation. Analytical Method
The High-performance liquid chromatography series of 1260 Infinity II equipped with Diode Array Detector HS (DAD), Infinity flexible pump, and 1260 Infinity II vial sampler, was used. The Data was processed using the ------- acquisition system. The chromatographic separation was performed using a C18 -Gemini 3 mm (150 mm x 4.6 mm) column set at 40C using Acetonitrile: Milli Q Water ( 99%: 1%) as a mobile phase at a flow rate of 1.5 ml/min. The injection volume was set at 100ml, the analyte was eluted at 7.1 minutes and the run time was set at 15 minutes. The detection was carried out at 265 nm.
Mobile phase preparation
A 500 mL measuring cylinder added 500 ml freshly dispensed Milli-Q Water and filtered through a 0.2 m Nylon 47 mm Membrane membrane Filterfilter, degassed during sonication. Labelled and dated properly as mobile phase A/B.100% Acetonitrile filtered through 0.2 m Nylon 47 mm Membrane filter and degassed during sonication. Labelled and dated properly.
Sampling and extraction procedure
The contents of each capsule were directly emptied into a 10 ml volumetric flask. Using a 29G needle, a small hole was punctured from one end of the capsule and air was administered into the other end to allow its full contents to be emptied. A single-edge blade was then used to slice the capsule shell in half. Ethanol (HPLC grade) was used to rinse the remaining contents and the sample was diluted up to the volume. The sample was sonicated for 15 minutes, and vortex mixed for 5 minutes. The high-speed centrifuge was used at 7000 rpm for 15 minutes and after that, the supernatant layer was collected for the HPLC analysis which was filtered using a Nylon 47 mm Membrane Filter.
Dissolution
HPLC analysis and Data processing
Validation Parameters
Validation of the analytical method
Method validation is a process used to demonstrate an analytical method's suitability for an interesting purpose. According to ICH (International Conference on Harmonization), each analytical method needs to be validated before its use. A validation examination includes testing multiple attributes of a method to determine valid facts whilst used robotically. The following parameters were used to determine the analytical performance of the method ADDIN EN.CITE <EndNote><Cite><Author>ICH</Author><Year>2022</Year><RecNum>1232</RecNum><DisplayText>(ICH, 2022)</DisplayText><record><rec-number>1232</rec-number><foreign-keys><key app="EN" db-id="09s5sdvs6vwpaee250uxd9230vxdx5xepaxt" timestamp="1662020703">1232</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>ICH</author></authors></contributors><titles><title>Validation of analytical procedures</title><secondary-title>International Council for Harmonisation of technical requirements for human use 
</secondary-title></titles><dates><year>2022</year></dates><urls><related-urls><url>https://www.ema.europa.eu/en/documents/scientific-guideline/ich-guideline-q2r2-validation-analytical-procedures-step-2b_en.pdf</url></related-urls></urls></record></Cite></EndNote>(ICH, 2022).
System suitability
Carryover
Linearity
System precision
Accuracy
Solution stability
Limit of Detection (LOD)
Limit of Quantitation (LOQ)
System suitability
System suitability is conducted to ensure the reproducibility, efficiency, and reliability of the chromatographic system before analysis as well as during HPLC analysis and the acceptance criteria according to the ICH.
The system suitability test prepared six injections of standard solution A and two injections of standard solution B at 10 mg/L. Standard solution B (Quality Control) was used to check standard solution A. The HPLC condition parameters were the same for both standards. The condition parameters include 100ml injection volume, 265 nm detection wavelength, Mobile phase Acetonitrile: Milli Q water (99:1%), Oven temperature 40C, and the flow rate was set at 1.5 ml/min. The standard solutions of 10 mg/L were prepared from a stock solution of 100 mg/L by diluting 100ml of the stock solution into a 10 ml volumetric flask using methanol to dilute the standard. The standard solution was vortexed for a few minutes to enhance its homogeneity of the standard solution. System suitability showed repeatability of injections.
Linearity
A stock solutions concentration was prepared by accurately weighing 5 mg of the reference standard of Vit D3 into a 50 ml volumetric flask and diluting to the volume with methanol, sonicated for 10 minutes, and labelled as Linearity Stock Solution 1 (100mg/L). Stock solution 2 was prepared by taking 100 l of standard solution 1 into 10 ml of the volumetric flask, diluting to the volume using methanol. To confirm the linearity of the analytical method five different calibrations; 10, 5, 1, 0.5, and 0.1 mg/L were prepared from the stock solution.
Precision
According to the ICH guideline principle, injection precision is assessed by analysing the standard solution of 1mg/L six times under the same chromatographic condition ADDIN EN.CITE <EndNote><Cite><Author>ICH</Author><Year>2022</Year><RecNum>1232</RecNum><DisplayText>(ICH, 2022)</DisplayText><record><rec-number>1232</rec-number><foreign-keys><key app="EN" db-id="09s5sdvs6vwpaee250uxd9230vxdx5xepaxt" timestamp="1662020703">1232</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>ICH</author></authors></contributors><titles><title>Validation of analytical procedures</title><secondary-title>International Council for Harmonisation of technical requirements for human use 
</secondary-title></titles><dates><year>2022</year></dates><urls><related-urls><url>https://www.ema.europa.eu/en/documents/scientific-guideline/ich-guideline-q2r2-validation-analytical-procedures-step-2b_en.pdf</url></related-urls></urls></record></Cite></EndNote>(ICH, 2022). The precision injection can determine the instruments sensitivity and identify the instrument's error during the instruments run. Standard solution of 1mg/L prepared from the stock solution of 100mg/L by diluting 100 ml in 10 ml volumetric flask.
Accuracy
Accuracy was performed by preparing three stock solutions of 100 mg/L, accurately weighing 5 mg into a 50 ml volumetric flask, and diluting the sample with methanol. The nominal concentrations at 40, 100, and 120 % were prepared in triplicates from stock solutions. The recovery percentage was calculated using the linearity equation y=69.686x-0.8616.
LOD and LOQ
The limit of detection is the lowest concentration of analyte which can be detected by the instrument at a 3:1 ratio of signal to noise and the limit of quantitation is the lowest concentration of the analyte which can be quantified by the instrument at a 10:1 ratio of signal to noise. For the determination of signal to noise a range of standard solutions from 0.1 to 0.6 mg/L was prepared and run under chromatographic conditions. The standard solutions of 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/L were prepared from stock solution of 100mg/L. For this series of standard solutions, first, dilute 100 l of stock dilution into a 10 ml volumetric flask to make a 1mg/L, then a series of standard solutions were prepared accordingly using methanol to dilute the standard solutions.
Results and discussion
System suitability:
The percentage drift peak and retention time were calculated following the below formula.
% Drift Peak area = (PA of standard 1 Peak area of QC) / PA of QC X100
% Drift Peak area= (66.458-65.827)/65.827*100=0.96%
% Drift Retention time= (RT standard 1 RT of QC) / RT of QC X100
%Drift retention time= (7.1655-7.1625)/7.1625*100=0.042%
Table 1 Results from standard solution A (1mg/L) in six replicates.
Std A Peak rea Retention time(min) Theoretical plate Tailing factor Peak area
%RSD RT
%RSD Peak area
%drift RT
%drift
Injection 1 65.087 7.164 1.1% 0.07% 0.96% 0.042%
Injection 2 66.745 7.175 Injection 3 66.939 7.166 Injection 4 66.77 7.161 Injection 5 65.943 7.167 Injection 6 67.265 7.166 Table 2 Results from standard solution B (1 mg/L)in two replicates.
Std B Peak rea Retention time(min) Theoretical plate Tailing factor Peak area
%RSD RT
%RSD Peak area
%drift RT
%drift
Injection 1 65.975 7.158 0.32% 0.09% 0.96% 0.042%
Injection 2 65.679 7.167
Carryover
Extraction solvent (methanol) was injected after stock solution standard at a concentration of 100mg/L and there was no carryover detected.
Linearity
In order to do linear regression analysis, the results were first normalized. The calculation of the results in the form of Peak Area is calculated and the results are plotted against concentration mg/L to perform linear regression, the plot for Vit D3 is shown in Figure1.
The linear equation obtained from the calibration curve is y=69.686x-0.8616 and the correlation coefficient is R2=0.9991. Thus, the method passes all acceptance criteria set out according to ICH guidelines, the method showed a linear relationship between the concentrations of Vit D3 with the integral response (i.e. peak area) detected by the instrument.
Table 3 Linearity pipetting amount at 5 different points from 0.1 to 10 mg/L.
Concentration 100 mg/L 10 5
Volume of Stock Solution 1 (l) 900 950
Concentration 1 mg/L 1 0.5 0.1
Volume of Stock Solution 2 (l) 0 500 900
Figure 1- Linearity for Vit D3 is shown in the plot, which has a slope of 69.686 an intercept of -0.8616 and an R2 of 0.9991, meeting the acceptance criteria. Mobile phase Acetonitrile: Water (99:1%), flow rate 1.5 ml/min, detection 265 l , temperature held at 40 C, injection volume of 100ml, Gemini column 3mm C18 110 A, 150 x 4.6mm
Injection Precision
Injection precision was performed by analysing standard solutions six times under the chromatographic condition. The results from the precision injection indicated the RSD% for peak area and retention time were 0.57 % and 0.14 % respectively.
Table 4 injection Precision results
Concentration 1 mg/L Peak Area Retention time (min)
Injection 1 68.485 7.188
Injection 2 69.621 7.193
Injection 3 69.597 7.205
Injection 4 69.139 7.178
Injection 5 69.443 7.18
Injection 6 69.028 7.177
Accuracy
The relative standard deviation values were found to be within the ranges of ___, with the accuracy values of ____. The results indicated the accuracy values within +- 15%, which is consistent with the acceptable concentration range.
Table 5 Accuracy results
Concentration mg/L Peak Area Retention time(min) Tailing factor Number of Theoretical Plates Recovery %
40
A
B
C 100
A
B
C
120
A
B
C Standard solution stability
The stability study of the stock solution in methanol at room temperature was evaluated and the results are tabulated shown in table6Table 6. According to the data, the stability of the stock solution (100mg/l) dropped by 4.9% in 48hours at room temperature.
Table 6 Standard solution stability at room temperature- covered in foil.
Time (hour)
At room Temperature Peak Area Retention time (min) Theoretical plate number Tailing factor % Changes in peak area
0 738.282 6.958 0
24 723.754 7.286 1.9%
48 702.100 7.129 4.9%
LOD, LOQ
The LOD and LOQ result was calculated with the help of linearity equation y=69.686x-0.8616 and using the equation below:
LOD=3.3 x STD slope
LOQ=10 x STD/slope
The results indicated that the limit of detection and limit of quantitation were 0.5 and 1.5 mg/L respectively.
Table 7 LOD and LOQ results
Concentration (mg/L) Peak Area Retention time (min) 0.1 8.550 7.158 LOD= 0.5
LOQ=1.5
0.2 11.526 7.148 0.3 17.056 7.149 0.4 23.376 7.159 0.5 29.772 7.162 0.6 35.353 7.169 HPLC assay
result
Dissolution
Dissolution measures the extent of solution formation to determine the therapeutic effectiveness and bioavailability of the drug. Each dissolution beaker consisted of 350mL of 0.1 molar HCl buffer and was heated to 37 degrees Celsius. One capsule from each batch number was added to each beaker and stirred at 75 rpm. At specific time intervals of 10, 20, 30 and 45 minutes. 5ml of the solution was withdrawn and replaced with 5mL of the buffer solution. The following equipment used for the dissolution test ________?
Results to be finalised???
Force degradation
result
Friability
As anticipated, two different batches of the same brand passed the friability testing with <1% weight loss. A sample of whole tablets that corresponded to approximately 6.5g. Tablets were weighed and dedusted, Batch A resulted in a value of 6.7049g, Batch B resulted in a total value of 6.7191. Tablets from each batch were then placed in the drum (radius, diameter, depth?). At a 25rpm, tablets were allowed to rotate for 100 revolutions.
Batch A had a 0.02% loss in weight, Batch B had a 0.09% loss in weight. (This may be subjected to inaccuracy of weight??)
Considering the availability of different Vitamin D variations, this formulation is expected to withstand durability. Tablet formulations of Vitamin D supplements are not made to be crushed or chewed but swallowed whole, given the weight of the tablets before and after the friability test measured almost identical results.
Disintegration
As per ICH standards, all Vitamin D capsules ruptured permitting the escape of its contents and Vitamin D tablets disintegrated into small granules. During the test, 6 capsules/tablets were randomly selected from each batch number. Capsules and tablets were rested on a gauze mesh accompanied by discs to keep products from floating out of the test tubes. Disintegration took place in a liquid medium containing Millipore water at 37 degrees Celsius. The time it took for each individual capsule/capsule to disintegrate was recorded.
Results to be continued.....
Mass variation
20 capsules of each batch number were selected and individually weighed. The contents of each capsule were removed as applied in the extraction method and washed with a solvent such as ethanol to remove any remaining contents or residuals from the capsule shell. Each emptied shell was dried and reweighed. Each net mass of the capsule was calculated by subtracting the mass of the shell from the respective gross mass.
Conclusion
Acknowledgment
Reference
ADDIN EN.REFLIST ICH 2022. Validation of analytical procedures. International Council for Harmonisation of technical requirements for human use
Appendix
