# Hippuric and Methyl Hippuric Acids in Urine (8301)

​HIPPURIC and METHYL HIPPURIC ACIDS in urine (1) (2) (3) (4)
hippuric acid 2-methyl hippuric acid 3-methyl hippuric acid 4-methyl hippuric acid
C6 H5 CONHCH2 CO2 H CH3 C6 H5 CONHCH2 CO2 H CH3 C6 H5 CONHCH2 CO2 H CH3 C6 H5 CONHCH2 CO2 H
METHOD: 8301, Issue 3
CAS:
RTECS: MR815000
Issue 1: 15 February 1984 Issue 3: 15 March 2003
Exposure to toluene and xylenes
hippuric acid: N-benzoylglycine 2-methyl hippuric acid: N-(o-Toluoyl) glycine 3-methyl hippuric acid: N-(m-Toluoyl) glycine 4-methyl hippuric acid: N-(p-Toluoyl) glycine
SAMPLING SPECIMENS:
495-69-2 42013-20-7 27115-49-7 27115-50-7
EVALUATION: PARTIAL
BIOLOGICAL INDICATOR OF: SYNONYMS: (1) (2) (3) (4)
FW: 179.18 193.20 193.20 193.20
8301
1. Pre-shift urine 2. End of shift urine after 2 days exposure
VOLUME:
Complete spot void
PRESERVATIVE:
A few crystals of thymol; store @ 4o C
SHIPMENT:
Pack in styrofoam shipper with bagged refrigerant, ship overnight
MEASUREMENT TECHNIQUE:
HPLC-UV DETECTION
ANALYTES:
(1) hippuric acid (2) 2-methyl hippuric acid (3) 3- & 4-methyl hippuric acids
EXTRACTANT:
Ethyl acetate
INJECTION VOLUME: 10 :L
SAMPLE STABILITY: CONTROLS:
WAVELENGTH:
254 nm
COLUMN:
Reverse phase (C18), run at 37o C
MOBILE PHASE:
84/16/0.025% (v / v / v) water / acetonitrile / glacial acetic acid; 1.5 mL/min
CALIBRATION:
Synthetic urine solutions of analyte.
RANGE:
10.0 to 1000 µg/mL.
ESTIMATED LOD:
(1) 4 µg/mL (2) 5 µg/mL (3) 6 µg/mL NOTE: See Method Evalution
PRECISION ( þ r ):
(1) 0.020 (see Table 1) (2) 0.015 (see Table 1) (3) 0.011 (see Table 1)
Stable 30 days @ 4o C Collect samples from unexposed, matched population.
APPLICABILITY: Hippuric acid and methyl hippuric acids are the principal metabolites of toluene and xylene, respectively. An occupational exposure to either of these organic solvents may be monitored by following the pattern of excretion of these metabolites in urine. Due to overlap in the range of hippuric acid concentration between exposed workers and non-exposed workers, monitoring urinary hippuric acid concentrations are better suited for groups of workers than for individuals [2]. INTERFERENCES: None known; however, para- and meta- isomers elute together in this system. There are other sources of hippuric acid such as food preservatives, ethyl-benzene, and styrene. OTHER METHODS: This is based on the method of Matsui, et al [3]. Method 8300 can be used for screening. Isotachophoresis has also been used [4]. More recent HPLC methodologies provide better resolution for these compounds [5].
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​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).
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 ​HIPPURIC and METHYL HIPPURIC ACIDS in urine: METHOD 8301, Issue 3, dated 15 March 2003 - page 3 of 5 6. Add 4 mL ethyl acetate. Mix for 2 minutes by rotation. 7. Centrifuge at 100 x gravity for 5 minutes.
NOTE:
r max = rotor radius Re lative Centrifu gal Force (R CF ) = gra vity 8. Transfer 200 µL of the organic (upper) layer to an HPLC vial and evaporate to dryness (- 30 minutes) using a heated (-30 oC) water bath and a gentle stream of nitrogen. 9. Re-dissolve the residue in 200 µL of distilled water.
CALIBRATION AND QUALITY CONTRO L: 10. Prepare working sta ndards over the ran ge of 1 0 to 1000 µg/m L by dilution of combined hippuric acids stock solution using synthe tic urine. W orking standards are stable for 1 week at room temperature and stab le for 30 da ys at 4 oC [1]. 11. Include control samples from an unex pos ed, m atch ed p opu lation with each an alytical run. If controls were not sent, use pooled, unexposed human urine. 12. Extract and analyze the working standards together with samples, and controls (steps 4 through 9, 14 and 15). 13. Analyze a dup licate field sample injection at a frequency of one replicate per 10 samples to monitor instrum ent ac curacy.
MEASUREMENT: 14. Set up the HPLC according to the manufacturer’s recomm endations and to conditions on page 8301-1. 15. Inject 10 µL of the standards, samples, and control samples into the HPLC. Determine peak heights.
CALCULATIONS: 16. Ca lculate the concentration of each analyte in the urine sample, C u (µg/m L), us ing the standard curve to correlate the peak heigh t with the con cen tration a m oun t. 17. Ca lculate the grams of analyte per grams of creatinine in the urine sample, C (g/g creatinine), using the concentration of an alyte in the urine sam ple, C u (µg/m L), and the concentratio n of creatin ine in urine dete rm ined in step 3, C R (g creatinine/L urine).
GUIDES TO INTERPRETATION: 1. Typical exc retion of h ippuric acid in non-exposed individuals is 1.0 g/g creatinine [2]. Meth yl hippuric acids are not found in non-exposed humans. 2. Th e T hres hold Lim it Value (TL V® ) [9] for u rine sam ples collected at the en d of a work shift is 1.6 g/g creatinine for hippuric acid a nd 1 .5 g/g crea tinine for m ethyl hipp uric acids.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​HIPPURIC and METHYL HIPPURIC ACIDS in urine: METHOD 8301, Issue 3, dated 15 March 2003 - page 4 of 5 EVALUATION OF METHOD: LOD/LOQ studies were p erform ed u sing standard s in synthetic u rine, standards in pooled urine, and standards in water. Standards dissolved in synthetic urine gave LODs of 4 µg/mL, 5 µg/mL, 6 µg/mL for hippuric acid, 2-methyl hippuric acid, 3-methyl hippuric acid, and 4-m eth yl hippuric acid, respectively. W ater proved to be a poor m atrix for hippuric acids, giving a high variability. Pooled urine also gave a high variability due to an initial background. Endogenous hippuric acids in pooled urine will skew the calibration curve. Reco very study data shows greater than 96% overall recovery for all analytes from spiked synthetic urine. Overall recovery rates were slightly lower (93% ) for spike d po oled urine, possibly due to variability in natu rally occurring hippuric acids. Precision, Accuracy, and Bias data (Table 1) were obtained for synthetic urine using 7 replica tes at 6 different c onc entra tion leve ls from 10 µ g/m L to 1000 µg/L. Results using pooled urine were similar. A 30-day stability study showed that the hippuric acids in synthetic urine were stable for 7 days at 22 oC and 30 days at 4 oC. In a single synthetic urine sample, hippuric acid was stable through temperature changes from -20 oC to 30oC for 24 hours. Sonication did not affect hippuric acid stability in the samples.
REFERENCES: [1] Mo tok GT , Perk ins JB, R eynolds JM [2002 ]. Hippuric and m ethyl hipp uric acids in urine, back up da ta report. Salt Lake City, Utah: DataChem Laboratories, Inc. [2] Pa cific T oxicology Laboratories [2003]. Hum an tox ic chem ical exposure, The Bulletin of P acific To xicology Labora tories, www.pa ctox .com /toluen e.htm . [3] Matsui H, Kasao M, Imam ura S [1978]. High performance liquid chromatographic determination of hippuric acid in hum an u rine, J. Chrom atog ., 45:231. [4] So llenberg J and Baldeste n A [1977]. Isota chophoretic analysis of m andelic acid, phenylglyoxylic acid, and/or xylen e, J. C hrom atog ., 132:469. [5] Ta rdif R and Brodeu r J [19 85]. J. Ana l. Tox ., 13:313. [6] Tietz NW [1976]. Funda m enta ls of C linical Ch em istry, 2 nd ed., pp . 994-999 , W .B. Sa und ers Co., Philadelphia, PA. [7] Lauwerys RR [1996]. Industrial chemical exposure: guidelines for biological monitoring. Davis, CA: Biomedical Publications, pp. 57-69. [8] Phipps F [1994]. Hippuric and methyl hippuric acids in urine, Method 8301, Issue 2. In: Eller PM, Ca ssinelli ME, eds. NIO SH Ma nua l of Analytical Metho ds, 4 th edition. Cincinnati, OH: Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health. DHHS (NIOSH) Publication No. 94-113. [9] UK [2003]. UK Government Information Notes on the Diagnosis of Prescribed Diseases. Conditions due to chem ical agent, www.m app erleyplains.co.uk /opru s/be nzen es.h tm.
METHOD UPDATED BY: George T. Motock, James B. Perkins, and John M. Reynolds, DataChem Laboratories, Inc., Salt Lake City, Utah 84123, under NIOSH contract CDC-200-2001-08000. Previous Issues Frederick C. Phipps, NIOSH/DBBS
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​HIPPURIC and METHYL HIPPURIC ACIDS in urine: METHOD 8301, Issue 3, dated 15 March 2003 - page 5 of 5 TAB LE 1 . OVE RAL L PR EC ISION , ACCUR ACY , AND BIAS Range Studied (µg/mL)
Estimated Accuracy (%)
Average
Hippuric acid
10-1000
16.0
-0.081
2-Methyl hippuric acid
10-1000
19.8
3-Methyl and 4-Methyl hippuric acids
10-1000
18.9
Compound
Bias
Overall Precision (ÖrT )
Instrument Precision (Sr)
-0.312-0.0237
0.0538
0.020
-0.099
-0.286-0.0141
0.0672
0.015
-0.097
-0.2959-0.3869
0.0615
0.011
Range
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition