Troubleshooting guide

VMA-SKREEN TROUBLESHOOTING GUIDE

PROBLEM	CAUSE	POSSIBLE REMEDY

1. Low Standard Curve)	(a) Insufficient mixing	(a) See General Problems 3b
	(b) Decomposed Standard	(b) After reconstitution, the standard must be refrigerated immediately after use. Prolonged contact with light or heat tends to cause decomposition.
	(c) No salt	(c) Check that 1.8 gms of sodium chloride is added in the first extraction step.
	(d) Inaccurate pipetting	(d) See General Problems 3c
	(e) VMA decomposition in Ethyl Acetate (pre-extraction step) which has not been promptly acidified.	(e) Immediately after the phases have separated transfer the pre-extraction Ethyl Acetate into test tubes containing 100 µl of Glacial Acetic Acid.
	(f) Contaminated Hydrochloric Acid and oxidant - (eg. nitric acid)	((f) A new bottle of reagent grade hydrochloric acid should be set aside for "VMA" procedure only.
	(g) Peroxide in Ethyl Acetate	(g) Test for Peroxide in Ethyl Acetate by Preparing a 50% Potassium Iodide Solution (5 gm in 10 mL of Deionized Water). The solution should be colorless. Add one mL of 50% KI to approximately 9 mL of Ethyl Acetate. Mix well. Any yellow coloration indicates Peroxide. Test several lots of Ethyl Acetate until you get one which is colorless or Peroxide free.

2. Low Control Value	(a) Insufficient mixing and extraction.	(a) See General Problems 3b
	(b) High pH of the specimen	(b) The pH of the urine specimen should be adjusted to below 3.0 during collection and storage. Higher pH will result in decomposition of VMA.
	(c) High urine pH during Ethyl Acetate extraction	(c) The pH of urine and controls should be below 2.0 during the 12 mL ethyl acetate extraction step.

3. High Control Value	(a) Low standard curve	(a) See VMA Steps 1a-1f
	(b) Ethyl Acetate not acidified with Glacial Acetic Acid following pre-extraction.	(b) Make sure that the Ethyl Acetate is properly acidified following the pre-extraction step. A non-specific orange color develops instead of the usual pink after addition of Diazo when the Acetic Acid is not added. This non-specific color may cause falsely elevated results.
	(c) Breakdown from exposure to light and/or heat	(c) VMA is light and heat sensitive. The standard, being colorless, breaks down more easily than the pigmented urine extracts.

4. Erratic Standard Curve and Control Value	(a) Erroneous pipetting	(a) See General Problems 3c
	(b) Inconsistent mixing	(b) The time and intensity of mixing from tube to tube should be consistent.

NOTE:
1. The reconstituted VMA standard is subject to decomposition when exposed to heat and light. As a check for decomposition we recommend freezing 3 mL aliquots of a freshly reconstituted and prepared 10 mg/L VMA standard. Aliquots should be thawed and run through the procedures weekly along with the regular standard curve. The absorbance of 10 mg/L VMA standard should read 210 ± 10. 2. It is preferred that the procedure be run without interruption. If however, the procedure must be interrupted, there are two possible steps where it may be done: (a) While washing solution is passing through the column. (10-15 min) (b) While 20 mL of water is passing through the column. (25-35 min)