Overview and Product System Choice

Two milliliters of acetonitrile was added into a plain tube containing 1ml of serum. This was then vortex mixed and centrifuged at 3000 r.p.m for 5 minutes. The clear supernatant was separated into a micro vial (Eppendorf) and stored at -20 degrees centigrade until when used.

7.5.3 Preparation of mixed standard (vitamin D2 and D3) concentrations in aqueous methanol for calibration

Stock: Vitamin D2 and D3 analytical standard concentration - 10mg/ml (10,000µg/ml) each.

From each vitamin D stock above, a 1 in 100 dilution with methanol was made to produce 100µg/ml concentration; this was further diluted (1 in 100) to produce a concentration of 1µg/ml (1000ng/ml). From the 1µg/ml solution, a 2 in 10 dilution was done to produce a concentration of 0.2 µg/ml (200ng/ml) of vitamin D2 and D3. Equal volumes of 200ng/ml solutions of vitamin D2 and D3 were then collected and mixed to get 100ng/ml of a mixed standard of both analytes.

The working standard concentrations of 10ng, 20ng, 40ng, 60ng and 80ng were prepared from the 100ng/ml standard concentration as shown below:

- 80ng/ml: 8ml of mixed standard (100ng/ml), made up to 10ml with methanol - 60ng/ml: 6ml of mixed standard (100ng/ml), made up to 10ml with methanol - 40ng/ml: 4ml of mixed standard (100mg/ml), made up to 10ml with methanol - 20ng/ml: 2ml of mixed standard (100mg/ml), made up to 10ml with methanol - 10ng/ml: 1ml of mixed standard (100mg/ml), made up to 10ml with methanol.

7.5.4 Preparation of mixed standard (vitamin D2 and D3) concentrations in serum for calibration

Stock concentration: 100,000ng/ml (100µg/ml). From the stock, different concentrations were prepared as shown in the table 1.

Table 1: preparation of vitamin D2 and D3 mixed standard concentrations in serum Mixed std

conc (ng/ml)

Volume of mixed std (ml)

Required conc (ng/ml)

Volume of serum (ml)

Volume of ACN (ml)

Total volume (ml)

1000 0.012 10 0.4 0.788 1.2

2000 0.012 20 0.4 0.788 1.2

4000 0.012 40 0.4 0.788 1.2

6000 0.012 60 0.4 0.788 1.2

8000 0.012 80 0.4 0.788 1.2

10,000 0.012 100 0.4 0.788 1.2

Mixed standard = mixed vitamin D2 and D3 analytical standards; ACN = acetonitrile

Table 2: Calibration plot for mixed concentrations of vitamin D2 and D3 in aqueous methanol Concentration

(ng/ml) in aqueous methanol

Vitamin D2

(Absorbance) m AU

Mean Vitamin D3

(Absorbance) m AU

Mean

10

20

40

60

80

100

0.28 0.29 0.21 0.34 0.25 0.17 0.13 0.14 0.54 0.57 0.52 0.43 0.42 0.39 1.05 1.01 0.97

0.26

0.29

0.146

0.54

0.41

1.01

1 0.9 0.9 13.38 7.38 1.44 1.43 1.44 18.14 18.15 17.99 22.15 22.0 22.11 70.79 70.77 70.6

0.93

10.38

1.436

18.09

22.09

70.72

m AU = milliabsorbance unit

7.5.5 TEST OF PURITY OF VITAMIN D3 TABLETS

Vitabiotics ultra D3 tablets were used for supplementation. The percentage purity was calculated by comparing the absorbance of equal concentrations of the drug with that of the analytical vitamin D3 standard.

Procedure for the preparation of 10,000ng/ml of vitabiotics ultra D3 tablets:

Each tablet of the drug contains 25µg (25,000ng) of cholecalciferol.

The weight of 10 tablets of vitabiotics ultra D3 tablets measured was 1.036g, with an average weight of 103.6mg. Hence 103.6 mg of the drug is equivalent to 25,000 ng of cholecalciferol. To 1 ml of N-hexane in a plain tube, 41.44 mg (equivalent to 10,000ng of cholecalciferol) of

crushed vitabiotics ultra D3 tablets was added, stirred and then centrifuged at 3000 r.p.m. The supernatant was then collected in a plain tube.

From the vitamin D3 analytical standard stock of 100, 000 ng/ml, a volume of 0.1ml was added to 0.9 ml of methanol to constitute 10,000 ng/ml. The spikes of both concentrations were the compared as shown on table.

Table: Test of purity of vitamin D3 tablets.

Concentration (ng/ml) Mean absorbance % purity Vitamin D3 tablets

(ultra D)

10,000 34.9

Vitamin D3 analytical standard

10,000 39.8

98.8

The purity of vitabiotics ultra D3 tablets was within acceptable limits for cholecalciferol as recommended by the British pharmacopoeia⁷⁹

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7.6 APPENDIX 6 PLASMA GLUCOSE ASSAY

Trinder’s analytical method was used. It uses glucose oxidase enzyme buffered in phenoxylate and dissolved in a coloured reagent. A solution constituted in 100mls containing: Potassium dihydrogen sulphate 38.9mls, Disodium hydrogen phosphate 61.1mls, Glucose oxidase 1.0ml, Phenol 0.64ml, 4-4aminophenaxone 20mg, sodium 100mg. The solution is stored at 4⁰C before use

Glucose Estimation

- 2ml of glucose oxidase containing solution was placed inside a test-tube.

- 2ml of plasma was added and the mixture is incubated at room temperature for 15mins - It was allowed to cool to room temperature during which time the colour changes from

colourless to pink depending on glucose concentration.

- The absorbance was read in a spectrophotometer at wave length of 540nm

- The reading was compared with the one of a standard glucose solution with a known glucose concentration which would have been incubated with the glucose oxidase solution.

Glucose in each sample will be estimated as follows:

Reading for sample x Amount in standard Reading for standard.

7.7 APPENDIX 7 7.7.1 GLYCATED HAEMOGLOBIN ASSAY

Test Principle (Boronate affinity chromatography):

Glycated proteins differ from non-glycated proteins by the attachment of a sugar moiety(s) at various binding sites by means of a ketoamine bond. Glycohemoglobin (GHb) thus contains 1, 2-cis-diol groups not found in non-glycated proteins. These diol groups provide the basis for separation of glycated and non-glycated components by boronate affinity chromatography. In this analytical technique, a boronate such as phenylboronic acid is bonded to the surface of the column support. When a solution of proteins (e.g. hemolysate) is passed through the column, the glycated component is retained by the complexing of its diol groups with the boronate. After the unretained non-glycated component elutes from the column and passes through the spectrophotometric detector, where it is detected at wavelength of 413 ±2 nm, the glycated component is eluted from the column with a reagent that displaces it from the boronate and then passes through the detector.

7.7.2 Test Procedure

1. The MiniPipette was filled with whole blood from the EDTA tube. The blood collected was then dispensed into the centre of the sample tray by pressing and releasing the plunger.

2. The flat end of the blood key was used to touch the blood drop, this allows it draw up blood by capillary action

3. Immediately, the blood key was introduced into the test cartridge through the blood port and firmly pressed until it locks in position.

4. Then the cartridge was immediately placed in the door of the analyzer. The OK button was then pressed to close the door and the test started.

5. After 10 minutes, the %HbA1c result was displayed, and the cartridge removed and disposed of.

7.7.3 CALCULATIONS

The glycated and non-glycated haemoglobin fractions were photometrically measured. The % HbA1c was then calculated from the absorbances using the following algorithm:

%HbA1c = M ( A glycated x 100 )+ C ( A glycated + A non-glycated)

Where M and C are slope and intercept factors to correct the values for DCCT calibration.

7.8 APPENDIX 8 SERUM INSULIN ASSAY

TEST PRINCIPLE

The insulin ELISA is a solid phase two-site enzyme immunoassay based on the sandwich technology, in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule. Insulin in the sample reacts with anti-insulin antibodies bound to micro-tiration wells and peroxidase-conjugated and anti-insulin antibodies in the solution.

ASSAY PROCEDURE

- Prepare sufficient microplate wells to accommodate calibrators and samples in duplicate - Pipette 25µL each of Calibrators, controls and samples into appropriate wells

- Add 100µL enzyme conjugate solution

- Incubate 1 hour on shaker at room temperature (18 to 25°C) - Wash plate six times with automatic plate washer

- Add 200µL substrate TMB

- Incubate 15mins. On shaker at room temperature

- Add 50µL Stop solution. Shake for approximately 5 seconds on shaker - Measure absorbance at 450nm and calculate results. Read within 30mins.