# Butoxyacetic Acid in Urine (8316)

​BUTOXYACETIC ACID IN URINE C6 H12 O3
MW: 132.16
8316
CAS: 2516-93-0
RTECS: None
METHOD: 8316, Issue 1
EVALUATION: PARTIAL
Issue 1: 15 March 2003
BIOLOGICAL INDICATOR OF: Exposure to
2-butoxyethanol 2-butoxyethyl acetate
SYNONYMS:
2-butoxyethanol:
Ethylene glycol monobutyl ether, Monobutyl glycol ether, Butyl Cellosolve®, Butyl oxitol, Dowanol® EB, EGBE, Ektasolve EB®, Jeffersol EB
2-butoxyethyl acetate:
Ethylene glycol monobutyl ether acetate, Butyl Cellosolve® acetate, Butyl glycol acetate, EGBEA, Ektasolve EB® acetate,
(CAS # 111-76-2, RTECS # KJ8575000) (CAS # 112-07-2, RTECS # KJ8925000).
SAMPLING
MEASUREMENT
SPECIMEN:
Urine
TECHNIQUE:
GAS CHROMATOGRAPHY, ECD Ni-63
VOLUME:
20 mL of sample
ANALYTE:
Pentafluorobenzyl butoxyacetate, PFB-BAA
PRESERVATIVE: None SHIPMENT:
Frozen; on dry ice
SAMPLE STABILITY:
At least 9 months at -70 /C
CONTROLS:
Urine specimens from non-exposed persons, number determined by design of study.
INJECTION VOLUME: TEMPERATURE -INJECTION: -DETECTOR: -COLUMN:
5 :L
150 /C 177 /C 70 /C for 2 min, 50 /C/min to 120 /C, 2 /C/min to 170 /C
CARRIER GAS:
Helium, 10 mL/min
COLUMN:
Capillary, fused silica, 5 m x 0.53-mm ID, deactivated and uncoated, followed by 30 m x 0.53-mm ID fused silica capillary with a 2.65-µm film of polydimethyl siloxane, HP-1 or equivalent.
CALIBRATION:
Standard solutions of PFB-BAA in toluene/2-propanol
QUALITY CONTROL:
Standard solutions of butoxyacetic acid (BAA) in urine
RANGE:
10 to 450 :mol/L in urine
ESTIMATED LOD:
10 :mol/L in urine
PRECISION ( þ r ):
0.13
ACCURACY:
Not determined
APPLICABILITY: Urinary butoxyacetic acid (BAA) is a biomarker of exposure to 2-butoxyethanol and 2-butoxyethyl acetate. Both 2-butoxyethanol and 2-butoxyethyl acetate are metabolized to butoxyacetic acid (BAA) and N-butoxyacetylglutamine, which are excreted in urine [1]. Since BAA produces the adverse hematogic effects attributed to exposures to 2-butoxyethanol and 2-butoxyethyl acetate, urinary BAA serves also as a biomarker to these particular exposure-related adverse health effects [2]. INTERFERENCES: No analytical interferences found. Consumption of ethanol is predicted to inhibit metabolism of 2-butoxyethanol to BAA [2], and thus may effect the accuracy of biomonitoring. OTHER METHODS: This method is based on those of Smallwood et al. [3] and Johanson [4]. Grosenken et al. [5] published a method using lyophilization, derivatization with pentafluorobenzyl (PFB) bromide, then GC. The method of Rettenmeier et al. [1] determined both free BAA and its conjugate with glutamine using extraction and derivatization with 4-nitrobenzyl bromide, then HPLC. Sakai et al. [6] used acid hydrolysis, extraction, derivatization with trimethylsilyldiazomethane, then GC to determine free plus conjugated BAA.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​BUT OX YAC ETIC IN UR INE: ME TH OD 8316, Issue 1, dated 1 5 Ma rch 2003 - Page 2 of 5 REAGENTS:
EQUIPMENT:
1. Te trabu tylam m onium hydrogen sulfate (C 16H 36NAHSO 4). 2. Potassium dihydrogen phosphate. 3. Potassium hydroxide. 4. Phosp horic acid, 85% .* 5. W ater, deionized. 6. C 16H 36NAHSO 4 in phosphate buffer: 0.2 M KH 2PO 4 and 0.1 M C16H 36NAHSO 4 in deionized water, made pH 6 with 85% H 3PO 4 or 10 M KOH. 7. Me thylene chloride.* 8. PF B brom ide, 2,3 ,4,5 ,6-penta fluorobenzyl brom ide.* 9. 2-Propa nol.* 10. To luene.* 11. Isopropano l-toluene m ixture, 1:1 (v/v). 12. PFB-BAA, pentafluorobenzyl ester of buto xyacetic ac id, MW 312 .24 am u. Synthesized ac cording to Groes ene ken [5], purified by RP-HPLC, and analyzed to one m ajor p eak by GC/M S. Dens ity 1.359 g /m L GC-MS (EI) fragmentation pattern; 240, 181, 131, 87, 73, 51amu. 13. PFB-BAA stock standard solution, 680 :mol/L. Dissolve 21 ± 0.1 mg of PFBBAA in 100 mL of 1:1 isopropanol-toluene using an am ber 1 00-m L volum etric flas k. Store at 0 to 5 / C. 14. BAA, Butoxyacetic acid, >99%. 15 Urine from une xpo sed volunteers , store d at -20 to -15 oC for a maximum of 2 weeks. 16. BAA-in-urine stock solution, 4000 :m ol/L. Dissolve 53 ± 1 mg of BAA in 100 mL of urine. Dilute with urine from unexposed volun teers for prepa ration of the qua lity control samples. 17 Argon with 5% methane. 18 Helium.
1. Gas chromatograph with 63Ni electron capture detector, temperature programm ing, splitless injection port with purge, and autoinjector. The detector makeup gas was 5% m ethane in argon at 60 mL/m in. 2. Polypropylen e bo ttles, 30- and 250 -m L with screw caps. 3. Graduated cylinders, 100- and 250-mL. 4. Shipping con tainer, polystyren e foa m , with dry ice. 5. Culture tubes, 16-mm x 100-mm , with teflonlined screw caps, disposable. 6. Disposable serological pipettes , 0.2 -m L in 0.001-m L increm ents, and 2-m L in 0.1-mL increments. Micropipette, 10-:L. 7. Alu m inum foil. 8. Tub e rotator. 9. Centrifuge, 3000 rpm. 10. Evaporator, nitrogen purge-type, with 30 / C heating bath. 11. Vortex m ixer. 12. Ultrasonic water bath, room tem perature. 13. Autoinjector vials, amber with crimp caps. 14. Ba lance, five -decim al-place analytic al. 15. Volumetric flasks, amber, 10- and 100-mL.
SPECIAL PRECAUTIONS: See material safety data sheets. Urine samples may contain a number of bacte rial and viral agents, including hepatitis B virus, a nd should be handled using Biosafety Level 2 prac tices, containm ent equipm ent and fa cilities [CD C & NIH , Biosafety in Microbiological and Biomedical Laboratories, 3rd ed., H HS P ublication No . (C DC) 93 -8395 (1 993)]. Pe nta fluorobenzyl bromide is volatile compound, a powerful lacrimator, and a suspected carcinogen. Methylene chloride, isopropanol, and toluene are NFPA level 3 fire hazards. Phosphoric acid is highly corrosive.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​BUT OX YAC ETIC IN UR INE: ME TH OD 8316, Issue 1, dated 1 5 Ma rch 2003 - Page 3 of 5 SAMPLING: 1. Collect a spot urine sam ple in one or several 250-mL polypropylene bottles. Measure and record the volume of the whole voiding and transfer ap proxim ate ly 20 mL to a 30-mL polypropylene bottle. Label the bottle with the code unique to that specimen. Freeze imm ediately with dry ice. 2. Ship in a polystyrene-foam shipping container kept frozen with dry ice. 3. Store the samples at -76 °C until time of analysis.
SAMPLE PREPARATION: 4. Th aw th e urine sa m ple to ro om tem pera ture. R em ove a portion for creatinine ana lysis [8]. 5. Transfer 0.200 mL of the sample to a culture tube. 6. Add 1.80 mL of tetrabutylamm onium hydrogen sulfate in phosphate buffer, 2.00 mL of methylene chloride, and then 10 :L of pentafluorobenzyl bromide. Cap the tube. 7. W rap the tube with aluminum foil to prevent exposure to light, and tumble with the rotator for 20 hr at 30 rev/m in. Tim ing is critical, bec aus e an alyte rec overy reac hes a m axim um at 20 hr then de crease s. 8. At 20 hours, centrifuge culture tubes for 5 min at 3000 rpm. 9. Transfer 1.5 mL of the methylene chloride (lower layer)to a clean culture tube. Evapo rate to dryness under nitrogen at 30 / C. 10. Add 1.00 mL of 1:1 isopropanol-toluene to the residue, vortex for 1 min, then sonicate for 1 min. 11. Transfer the solution to an auto inje cto r vial, cap, and seal.
CALIBRATION AND QUALITY CONTRO L: 12.
13.
14.
15.
Calibrate the GC for each batch of samples. a. Prepare 9 working standards by diluting the PFB-B AA stoc k solution with 1:1 toluen e/pro pan ol to 9 equally-spaced concentrations between 0.8 and 68 :mol/L (equivalent to urina ry levels of 5.3 to 453 :m ol/L). b. Analyze these standards with the unknowns in step 17 in random order but after every third unknown in the batch. c. Prepare a calibration gra ph of p eak a rea versus analyte concentratio n, using, if necess ary, quadratic regression to fit the data. Prepare and analyze a minimum of 4 BAA-in-urine quality-control samples per batch. a. Prepare control samples at 0, 20, 90, and 400 :mol/L by diluting aliquots of BAA-in-urine stock solution with urine from unexposed individuals. b. Analyze quality control samples and five samples of the unspiked urine with the unknowns. c. Correct the nom inal values for the control samples for the background level of BAA in the blank urine. d. Calculate the recoveries and plot them on a control chart for the method. Re-analyze two field samples from the previous batch with each batch. a. If possible, select field sam ples with levels at both ends o f the analytical range, but above the dete ction lim it. b. Calculate the percent difference for each duplicate and plot it on a control chart for the method. Analyze at least one sample of pure water with each batch as a check for background interferences.
MEASUREMENT: 16. 17.
18.
Set the gas chrom atograph system according to the manufacturer’s recomm endations and the conditions given on page 8316-1. Inject 5 :L of sample from step 11 or standard from step 12. Note: W ith the chrom atog raph ic system use d for m etho d de velop m ent, the PFB-B AA peak had an efficiency of 124,000 plates, an asymm etry factor of 1.4, and a retention time of 31.7 min. The inten sity and precision of the detector’s response to PFB-BAA were sensitive to temperature, with an optimal precision obtained at 177 / C. Measure peak area.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​BUT OX YAC ETIC IN UR INE: ME TH OD 8316, Issue 1, dated 1 5 Ma rch 2003 - Page 4 of 5 CALCULATIONS: 19. 20.
Calculate from the calibration curve the co ncentration of PFB -BAA (C c, :mol/L) in the final solution. Calculate the conce ntration of BAA (C u, :m ol/L) in the urine sam ple fro m the equa tion be low.
21.
where 6.67 is the dilution for the sample preparation steps. Adjust the result for the creatinine level and report in micromoles per mole creatinine or in milligrams per gram creatinine, using the conversion 1 :mol/mol equals 1.17 mg/g.
GUIDES FOR INTERPRETATION: A urinary BAA level of 51 mm ol/mol creatinine (60 mg/g creatinine) corresponds to the NIOSH recomm ended expos ure level for a 10-hr work shift of 5-ppm TW A for 2-butoxyethanol and 2-butoxyethyl acetate, assuming no derm al exp osu re to the liquid. Biological monitoring for exposure is recomm ended, because 1) dermal absorption m ay be a m ajor ro ute of e xposure, 2) worklo ad can significantly influence inhalation exposure, and 3) bu toxyacetic acid itself exerts h em atolog ic toxicity [2]. BAA levels up to 282 :m ol/m ol creatinine (3 30 m g/g creatinine) have been m easured in occupation ally exposed persons [7]. Levels as high as 0.5 :m ol/m ol creatinine (0.6 m g/g creatinine) have been fou nd in urine of p eople not occupationally exposed to 2-butoxyethanol or 2-butoxyethyl acetate [6]. 2-Butoxyethanol is comm only found in household products, such as paints and cleaners.
EVALUATION OF METHOD: A detection limit of 10 :mol/L was estimated using resu lts from 11 urine samples spiked with BAA over the range 0.27 to 32 :m ol/L. Recovery averaged 93% (range 53% to 146%) for 20 quality-control samples con tain ing BAA at concentrations 12 to 3000 :m ol/L. Precision was estimated a t 13% relative standard deviation from duplicate analysis of 12 field sam ples from workers exposed to 2-buto xyetha nol.
REFERENCES: [1]
[2] [3] [4] [5] [6]
Rettenmeier AW , Hennigs R, W odarz R [1993]. Determination of butoxyacetic acid and n-butoxyacetylglutamine in urine of lacquerers exposed to 2-butoxyethanol, Int Arch Occup Environ He alth, 65, S151-S153. NIOSH [1990]. Criteria for a Recom mended Standard. Occupational exposure to ethylene glycol m onobutyl ether and ethylene glycol monobutyl ether acetate, DHHS (NIOSH) Publication No. 90-118. Sm allwood AW , DeBord K, Burg J, Moseley C, Lowry L [1988]. D ete rm ination of u rinary 2-e tho xyacetic acid as an indicator of occupational exposure to 2-ethoxyethanol, Appl. Ind. Hyg., 3(2), 47-50. Johanson G [1989]. Analysis of ethylene glycol ether metabolites in urine by extractive alkylation and electron-capture gas chrom ato graphy, A rch. T oxicol., 63, 107-111. Groesenken D, Veulem ans H , Massc helein R, Van Vlem E [1989]. An imp roved method for the determination in urine of alkoxyacetic acids, Int. Arch. Occup. Environ. Health, 61, 249-254. Sakai T, Araki T, Morita Y, Masuyama Y [1994]. Gas chromatographic determination of butox yacetic acid after hydrolysis of conjugated metabolites in urine from workers ex pos ed to 2-bu toxyethan ol, Int. Arc h. O ccu p. En viron. H ealth, 66, 249-254.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​BUT OX YAC ETIC IN UR INE: ME TH OD 8316, Issue 1, dated 1 5 Ma rch 2003 - Page 5 of 5 [7] [8]
Ke lly JE, Van Gilder TJ [1994]. Health hazard evaluation rep ort no. HET A-93-0562-2464, Ohio University, Athens, Ohio. Cincinnati, OH: National Institute for Occupational Safety and Health. Spencer K [1986]. Analytical reviews in clinical biochemistry: The estimation of creatinine, Ann. Clin. Biochem, 23, 1-25.
METHOD DEVELOPED BY: Kenneth K. Brown Ph.D., NIOSH/DART
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition