HIPPURIC and METHYL HIPPURIC ACIDS in urine: METHOD 8301, Issue 3, dated 15 March 2003 - page 2 of 5
EQUIPMENT:
REAGENTS: 1. 2. 3. 4. 5. 6. 7. 8. 9.
10.
11. 12. 13.
Thymol, USP. Sodium chloride. Hydroch loric ac id (6N ).* Ethyl acetate, HPLC grade. Hipp uric acid, reage nt gra de. 2-Methyl hippuric acid, reagent grade. 3-Methyl hippuric acid, reagent grade. 4-Methyl hippuric acid, reagent grade. Co m bined hipp uric acids stoc k solution, 1 mg/m L per analyte. Add 35 mg each of hippuric acid, 2-m eth yl hippuric acid, 3-m eth yl hippuric acid, and 4-methyl hippuric acid to 35 m L synth etic urin e in a 40-m L vial. C ap tightly and sonicate for 30 min, then place in 30 oC water bath for 5 m in. Visually inspec t to verify that all compounds are in solution. NOTE: T he hippuric acid solution will precipitate out at room tem perature, s o this preparation should be used imm ediately after removal from the 30 oC water bath. Mo bile phase . Add 840 m L distilled w ater to 160 mL acetonitrile (HPLC grade) and 250 µL of glacial acetic acid. Mix and filter. Pre-purified nitrogen, 99.9%. Synthetic urine (Uri sub™ from CST Tec hnologies, Inc., Great Neck , NY). Pooled, unexpo sed , hum an u rine [1].
1. Bottles, polyethylene, 250-mL. 2. Refrigerant, bagged (“Blue Ice,” or equivalent). 3. HPL C system consistin g of sam ple injector, pump, ultraviolet detector at 254 nm, strip chart recorder, integrator, RP C18 column, and column heater (Supelco Discovery #322 201-01 or eq uivalent). 4. Heated water bath purged by nitrogen blow-down apparatus. 5. Centrifuge. 6. Analytical balance. 7. Tu bes , 15-m L bo rosilicate glas s with caps. 8. HPLC vials and caps with limited volume inserts (Kimble Glass, Inc., Art. No. 60745N -1232). 9. Micro-syringes: 10-µL, 100-µL, 250-µL, 500-µL, 1-mL and 10-mL. 10. Positive displaceme nt pipette (40-µL). 11. Rotation Mixer (Fisher model 34601 or equivalent).
- See SPECIAL PRECAUTIONS
SPECIAL PRECAUTIONS: Sam ples of urine co llected from hum ans pos e a re al hea lth risk to laboratory workers who collect and handle these samples. These risks are primarily due to personal contact with infective biological samples and can have serious health consequences, such as infectious hepatitis, and other diseases. Pre-imm unization against Hepatitis C is highly recomm ended. Those who han dle urine sp ecim ens sho uld wear p rotec tive gloves, and a void aeros olization of the s am ples. Mo uth pipetting, of co urse, m ust be avoided. Acids are ex trem ely corro sive; w ork with the m in a fum e ho od a nd w ear p rotec tive safety equipm ent.
SAMPLING: 1. Collect pre-shift and post-shift urine in a 250-m L polyeth ylene bottle conta ining a few crysta ls of thym ol. NOTE: Take the sam ple at the en d of the se con d da y of suspe cted exp osu re to toluene or xylene. Also take pre-exposure samples and samples from non-exposed workers as controls. 2. Pack bottles in styrofo am shipp er with bag ged refrige rant a nd s hip overnight.
SAMPLE PREPARATION: 3. Perform a cre atinine determ ination on an aliquot of urine [6]. 4. Pipette 1.0 mL of well-mixed urine into a 15-mL borosilicate glass tube with cap. 5. Add 80 µL of 6 N Hcl with positive displacement pipette, mix, and add 0.3 grams sodium chloride.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition
