- A. MANUAL ASSAY PROCEDURE:
- B. AUTO-ANALYSER ASSAY PROCEDURE:
Reagent preparation is performed as follows:
- C. MICROPLATE ASSAY PROCEDURE:
NOTES:
1. The Microplate Assay Procedure for citric acid can be performed using either a single point standard or a full calibration curve.
2. For each batch of samples that is applied to the determination of citric acid either a single point standard or a calibration curve must be performed concurrently using the same batch of reagents.
- SAMPLE PREPARATION:
1. Sample dilution (for “manual format”).
The amount of citric acid present in the cuvette (i.e. in the 0.20 mL of sample being analysed) should range between 1.0 and 100 μg. The sample solution must therefore be diluted sufficiently to yield a concentration between 0.005 and 0.50 g/L.
2. Sample clarification:
Carrez reagents cannot be used for deproteinisation as their use results in significantly reduced recoveries. Perchloric or trichloroacetic acid are used as alternatives (see specific examples).
3. General considerations.
(a) Liquid samples: clear, slightly coloured and approximately neutral, liquid samples can be used directly in the assay.
(b) Acidic samples: if > 0.2 mL of an acidic sample is to be used undiluted (such as wine or fruit juice), the pH of the solution should be increased to approx. 7.4 using 2 M NaOH, and the solution incubated at room temperature for 30 min.
(c) Carbon dioxide: samples containing significant quantities of carbon dioxide, such as beer, should be degassed by increasing the pH to approx. 7.4 with 2 M NaOH and gentle stirring, or by stirring with a glass rod.
(d) Coloured samples: an additional sample blank, i.e. sample with no CL, may be necessary in the case of coloured samples.
(e) Strongly coloured samples: if used undiluted, strongly coloured samples should be treated by the addition of 0.2 g of polyvinylpolypyrrolidone (PVPP)/10 mL of sample. Shake the tube vigorously for 5 min and then filter through Whatman No. 1 filter paper.
(f) Solid samples: homogenise or crush solid samples in distilled water and filter if necessary.
(g) Samples containing fat: extract such samples with hot water at a temperature above the melting point of the fat, e.g. in a 100 mL volumetric flask at 60°C. Adjust to room temperature and fill the volumetric flask to the mark with water. Store on ice or in a refrigerator for 15-30 min and then filter. Discard the first few mL of filtrate and use the clear supernatant (which may be slightly opalescent) for assay.
(h) Samples containing protein: deproteinise samples containing protein by adding an equal volume of ice-cold 1 M perchloric acid with mixing. Centrifuge at 1,500 g for 10 min and neutralise the supernatant with 1 M KOH. Alternatively, use trichloroacetic acid.
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- 商品規格
72 assays (manual) / 720 assays (microplate) / 840 assays (auto-analyser)
Bottle 1:
Buffer (40 mL, pH 7.5) plus sodium azide (0.02%) as a preservative.
Stable for > 2 years at 4°C.
Bottle 2:
NADH plus PVP.
Stable for > 5 years below -10°C.
Bottle 3:
L-Malate dehydrogenase plus D-lactate dehydrogenase, 1.5 mL.
Stable for > 2 years at 4°C.
Bottle 4: *3
Citrate lyase lyophilisate.
Stable for > 2 years below -10°C.
Bottle 5:
Citric acid standard solution (5 mL, 0.20 mg/mL) in 0.02% (w/v) sodium azide.
Stable for > 2 years; store sealed at 4°C.
