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CLIN. CHEM. 41/2, 300-305 (1995) #{149} Drug Monitoring and Toxicology 300 CLINICAL CHEMISTRY, Vol. 41, No. 2, 1995 Simultaneous Determination of Alcohols and Ethylene Glycol in Serum by Packed- or Capillary-Column Gas Chromatography John F. Livesey,’ Sherry L. Perkins,” Nancy E. Tokessy,’ and Margaret J. Maddock’ We developed a packed-column chromatographic pro- cedure capable of simultaneous quantitation of metha- nol, ethanol, isopropanol, acetone, and ethylene glycol. This method was then updated to a rapid, sensitive, wide-bore capillary method. The packed-column system uses direct injection of 1 &L of Na2WO4/H2S04-depro- teinized serum onto a 1.8 m x 2 mm (i.d.) column packed with 80/100 HayeSep A. A linear temperature gradient from 90 to 205#{176}C allows complete elution of all compo- nents within 20 mm; minimum detection limits are 2 mmoVL. The wide-bore capillary method uses 0.1 &L of sample deproteinized by ultrafiltration, injected onto a 30 m x 0.53 mm (i.d.) 3-sm Rtx-200 (Restek) column. Baseline resolution to a minimum detection limit of 0.1 mmoVL of all compounds is achieved in 5 mm with a linear temperature gradient from 40 to 250#{176}C and dual internal standards of n-propanol and 1 ,2-butanediol. Indexing Terms: methanol/toxicology Diagnosis and treatment of intentional and acciden- tal poisonings after ingestion of methanol, isopropanol, or ethylene glycol require accurate, sensitive, rapid, and reliable methods of analysis. Previous chromato- graphic methods (1-7) have required separate columns and (or) chromatographic systems to analyze alcohols and diols, time-consuming and potentially incomplete derivatizations of ethylene glycol, and long analysis times. Enzymatic methods (8, 9) for these compounds have had only limited success and do not allow simul- taneous quantitation of all alcohols and diols. Here we describe two different single-run gas-chro- matographic (GC) methods for the simultaneous quan- titation of alcohols and diols, with minimal preanalyti- cal sample preparation.4 The first uses packed columns, with a time between injections of 20 mm. The second uses a wide-bore capillary column to attain >10-fold improvement in detection limits, with injec- tions possible every 10 mm. ‘Department of Laboratory Medicine, Ottawa Civic Hospital, 1053 Carling Ave., Ottawa, Ontario, Canada K1Y 4E9. 2 Department of Pathology and Laboratory Medicine, Univer- sity of Ottawa, Ottawa, Ontario, Canada. 3Address correspondence to this author, at the Ottawa Civic Hospital, Fax 613-761-5361. 4Nonstandard abbreviations: GC, gas chromatography(-ic); FID, flame ionization detector; and PEG, polyethylene glycol. Received July 5, 1994; accepted November 17, 1994. Materials and Methods Materials Chemicals. OmniSolv#{174}-grade methanol, acetone, 2-propanol, and sodium tungstate were obtained from BDH (Toronto, ON). Ethylene glycol (1,2-ethanediol), isobutyl alcohol, 1-butanol, isopentyl alcohol, 1-penta- nol, formaldehyde, chloroform, dichloromethane, aceto- nitrile, ethyl acetate, isopropylacetone, 2,4-pentanedi- one, cyclohexanone, glycerol, and dimethyl sulfoxide were from Fisher Scientific (Nepean, ON). Propionic acid, n-propanol, 1,2-butanediol, 1,2-propanediol, 1,3- propanediol, 2,3-butanediol, 1,3-butanediol, 2-methyl- 2,4-pentanediol, 1,4-butanediol, 2-butanone, 3-pen- tanone, 2-pentanone, and hydroxyacetone were from Aldrich (St. Louis, MO). Oxalic acid was from Sigma (St. Louis, MO). Ethanol was obtained from Commer- cial Alcohols (Brampton, ON), sulfuric acid from J. T. Baker (Phiuipsburg, NJ). Standards and quality-control materials. We pre- pared an aqueous stock solution (generally 10 milL) of each alcohol and diol in distilled, deionized water. This stock was used to prepare fortified serum standards from a serum pool confirmed (by GC) to be alcohol- and diol-free. Aliquots (1 mL) were frozen (-20#{176}C) in Nal- gene cryovials until needed. After thawing, these se- rum standards were treated the same as ordinary serum samples, with internal standards (n-propanol for alcohols, 1,2-butanediol for diols) added at the time of analysis. Packed-Column GC Analyses Instrumentation. A Varian Model 3700 gas chro- matograph (Varian Canada, Ottawa, ON) with dual columns and flame ionization detectors (FID) was used. The columns were 1.8 m X 2 mm (i.d.) packed with 80/100 HayeSep R (Supelco, Bellefonte, PA). The car- rier gas was nitrogen at 45 mllmin. Injector and FID temperatures were 220#{176}C; the oven temperature was programmed from 90 to 205#{176}C at 10#{176}C/mm, and then held at 205#{176}C for 2 miii. For diols other than ethylene glycol, the final temperature was increased to 235#{176}C. The 1-L injections were made manually, every 30 mm, with a 5-LL gas-tight syringe (Hamilton, Reno, NV). Detector output was monitored with a Recordall Series 5000 (Fisher Scientific, Nepean, ON) chart re- corder. Sample preparation. To deproteiize the serum! plasma samples and the fortified serum standards, we vortex-mixed 200 L of each serum, 100 .tL of internal standard (n-propanol, final concentration -25 mmol! L), 100 g/L Na2WO4, and 1.3 mmol/L H2S04 and then
Transcript
Page 1: Simultaneous Determination of Alcohols and Ethylene Glycol in ...

CLIN. CHEM. 41/2, 300-305 (1995) #{149}Drug Monitoring and Toxicology

300 CLINICAL CHEMISTRY, Vol. 41, No. 2, 1995

Simultaneous Determination of Alcohols and Ethylene Glycol in Serum by Packed-or Capillary-Column Gas ChromatographyJohn F. Livesey,’ Sherry L. Perkins,” Nancy E. Tokessy,’ and Margaret J. Maddock’

We developed a packed-column chromatographic pro-cedure capable of simultaneous quantitation of metha-nol, ethanol, isopropanol, acetone, and ethylene glycol.This method was then updated to a rapid, sensitive,wide-bore capillary method. The packed-column systemuses direct injection of 1 &L of Na2WO4/H2S04-depro-teinized serum onto a 1.8 m x 2 mm (i.d.) column packedwith 80/100 HayeSep A. A linear temperature gradientfrom 90 to 205#{176}Callows complete elution of all compo-nents within 20 mm; minimum detection limits are 2mmoVL. The wide-bore capillary method uses 0.1 &L ofsample deproteinized by ultrafiltration, injected onto a 30m x 0.53 mm (i.d.) 3-sm Rtx-200 (Restek) column.Baseline resolution to a minimum detection limit of 0.1mmoVL of all compounds is achieved in 5 mm with alinear temperature gradient from 40 to 250#{176}Cand dualinternal standards of n-propanol and 1,2-butanediol.

Indexing Terms: methanol/toxicology

Diagnosis and treatment of intentional and acciden-

tal poisonings after ingestion of methanol, isopropanol,or ethylene glycol require accurate, sensitive, rapid,and reliable methods of analysis. Previous chromato-graphic methods (1-7) have required separate columnsand (or) chromatographic systems to analyze alcoholsand diols, time-consuming and potentially incompletederivatizations of ethylene glycol, and long analysistimes. Enzymatic methods (8, 9) for these compoundshave had only limited success and do not allow simul-taneous quantitation of all alcohols and diols.

Here we describe two different single-run gas-chro-matographic (GC) methods for the simultaneous quan-titation of alcohols and diols, with minimal preanalyti-cal sample preparation.4 The first uses packedcolumns, with a time between injections of 20 mm. Thesecond uses a wide-bore capillary column to attain>10-fold improvement in detection limits, with injec-tions possible every 10 mm.

‘Department of Laboratory Medicine, Ottawa Civic Hospital,1053 Carling Ave., Ottawa, Ontario, Canada K1Y 4E9.

2 Department of Pathology and Laboratory Medicine, Univer-

sity of Ottawa, Ottawa, Ontario, Canada.3Address correspondence to this author, at the Ottawa Civic

Hospital, Fax 613-761-5361.4Nonstandard abbreviations: GC, gas chromatography(-ic);

FID, flame ionization detector; and PEG, polyethylene glycol.Received July 5, 1994; accepted November 17, 1994.

Materials and Methods

Materials

Chemicals. OmniSolv#{174}-grade methanol, acetone,2-propanol, and sodium tungstate were obtained fromBDH (Toronto, ON). Ethylene glycol (1,2-ethanediol),isobutyl alcohol, 1-butanol, isopentyl alcohol, 1-penta-nol, formaldehyde, chloroform, dichloromethane, aceto-nitrile, ethyl acetate, isopropylacetone, 2,4-pentanedi-one, cyclohexanone, glycerol, and dimethyl sulfoxidewere from Fisher Scientific (Nepean, ON). Propionicacid, n-propanol, 1,2-butanediol, 1,2-propanediol, 1,3-propanediol, 2,3-butanediol, 1,3-butanediol, 2-methyl-2,4-pentanediol, 1,4-butanediol, 2-butanone, 3-pen-tanone, 2-pentanone, and hydroxyacetone were fromAldrich (St. Louis, MO). Oxalic acid was from Sigma(St. Louis, MO). Ethanol was obtained from Commer-cial Alcohols (Brampton, ON), sulfuric acid from J. T.Baker (Phiuipsburg, NJ).

Standards and quality-control materials. We pre-pared an aqueous stock solution (generally 10 milL) ofeach alcohol and diol in distilled, deionized water. Thisstock was used to prepare fortified serum standardsfrom a serum pool confirmed (by GC) to be alcohol- anddiol-free. Aliquots (1 mL) were frozen (-20#{176}C)in Nal-gene cryovials until needed. After thawing, these se-rum standards were treated the same as ordinaryserum samples, with internal standards (n-propanolfor alcohols, 1,2-butanediol for diols) added at the timeof analysis.

Packed-Column GC Analyses

Instrumentation. A Varian Model 3700 gas chro-matograph (Varian Canada, Ottawa, ON) with dualcolumns and flame ionization detectors (FID) was used.The columns were 1.8 m X 2 mm (i.d.) packed with80/100 HayeSep R (Supelco, Bellefonte, PA). The car-rier gas was nitrogen at 45 mllmin. Injector and FIDtemperatures were 220#{176}C;the oven temperature wasprogrammed from 90 to 205#{176}Cat 10#{176}C/mm,and thenheld at 205#{176}Cfor 2 miii. For diols other than ethyleneglycol, the final temperature was increased to 235#{176}C.The 1-L injections were made manually, every 30mm, with a 5-LL gas-tight syringe (Hamilton, Reno,NV). Detector output was monitored with a RecordallSeries 5000 (Fisher Scientific, Nepean, ON) chart re-corder.

Sample preparation. To deproteiize the serum!plasma samples and the fortified serum standards, wevortex-mixed 200 L of each serum, 100 .tL of internalstandard (n-propanol, final concentration -25 mmol!L), 100 g/L Na2WO4, and 1.3 mmol/L H2S04 and then

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1000

800

600

400

200

CLINICAL CHEMISTRY, Vol. 41, No. 2, 1995 301

centrifuged at 13 000g for 2 mm. The supernates weredecanted and retained for injection.

Capillary-Column GC Analyses

Instrumentation. We used an HP 5890A GC(Hewlett-Packard, Mississauga, ON) with an un-purged, packed-column injection port modified for usewith a capillary column. For the inlet conversion weused a deactivated fused-silica Uniliner#{174}injection-portliner and sleeve adaptor (Restek, Bellafonte, PA). Thecarrier gas was helium at a linear velocity of 100 cm/s(-18 mL/min at 250#{176}C,column head-pressure -345kPa). Nitrogen make-up gas (30 mllmin) was used atthe detector, with FID sensitivity set at 4-8 x i0 Afull-scale, and temperature set at 250#{176}C.Detector out-put was monitored with a Varian 4290 integrator(Varian Canada) or an ACI chromatography worksta-tion (Dionex, Mississauga, ON).

All three columns tested were 30 m X 0.53 mm (i.d.),obtained from Chromatographic Specialties (Brock-ville, ON): (a) 5 m DB-1; (b) 3 im DB-624 (both fromJ & W Scientific, Folsom, CA); and (c) 3 m Rtx-200(Restek). A 5-m length of 0.53 mm (i.d.) deactivatedfused-silica tubing was used as a retention gap/guardcolumn in each case.

Direct injection of 0. 1-L samples was performedmanually with a Model 18O1RN (Hamilton) gas-tightsyringe with a 5-cm blunt-tip needle, through Thermo-lite#{174}(Restek) high-temperature septa. For automatedinjections (0.1 L) we used an HP 7673B (Hewlett-Packard) autoinjector with nanoliter adaptor through a5-L syringe with a 23/26-gauge tapered, removableneedle (Hewlett-Packard).

The optimized oven temperature program for theRtx-200 column was as follows: 40#{176}Cfor 1.0 mm,increase to 250#{176}Cat 70#{176}/mm,and hold for 2 min. Therun time was 5.0 mm, and the total time betweeninjections was -10 mm, including column cool-downand a 1-mm equilibration at 40#{176}C.For the DB-1 andDB624 columns, the conditions were as follows: 125#{176}Cfor 2 min, increase to 250#{176}Cat 50#{176}/mm,and hold for 2mm.

Sample preparation. To deproteinize serum/plasmasamples and the fortified serum standards, we pipetted100 L of serum plus 25 &L of internal standards (foralcohols, n-propanol; for ethylene glycol, 1,2-butane-diol; final concentrations, -25 mmol/L) into Ultra-free#{174}-MC(Millipore Canada, Mississauga, ON) ultra-filtration devices with 10 000-Da-cutoff regeneratedcellulose membranes preserved with triethylene glycol(not glycerol). The samples were centrifuged for 5 mmat 13 000g in a microcentrifuge, and the protein-freefiltrates were retained for analysis. For automatedinjections, we transferred the filtrate to 100-Lpolypropylene SnapCap GC#{174}autoinjector vials (SRi;Scientific Products and Equipment, Concord, ON).

Interference Studies

Interference by common therapeutic drugs wastested by analyzing a commercial therapeutic drug

monitoring control serum, Lyphocheck#{174}TherapeuticDrug Monitoring (TDM) Control Serum (Human) (Lev-el 3; Bio-Rad, Anaheim, CA). Other alcohols and diolswere investigated by analyzing serum supplementedwith each compound (at 5 mL/L).

Results

Representative chromatograms are shown in Fig. 1.The packed column and the Rtx-200 capillary columnattained baseline resolution between all the low-boil-ing-point alcohols and acetone. All compounds of inter-est were eluted within 3 mm. There were no coelutionproblems with compounds previously reported (10, 11)to interfere with ethylene glycol. In contrast, when weused the oven temperatures required for accurate mea-surement of diols, the DB-1 and DB-624 capillarycolumns were unable to separate acetone from isopro-panol, and the low-boiling-point alcohols were not base-line-resolved (chromatogram not shown). Retentiontimes are presented in Table 1.

Detection limits for all analytes were 0.1 mmol/L on

pA1000

800

600

400

200

3 4

MinutesFig. 1. (A-C) Capillary GC analyses of alcohols and diols on aRtx-200 column, 0.1-pt injection volume; (D) GC analysis onHayeSep 80/100 packed column, of alcohols and diols in 1.0 pi. ofthe calibrator shown in (A).Chromatographicconditions as described in Materials and Methods. 4)Serum calibrator, with analytesat 1 milL. Peak identities and concentrations:1, methanol 24.7 rnmoIIL; 2, ethanol 17.0 mmoVL; 3, isopropanol 13.1mmoVL; 4, n-propanol 21.4 mmoVL (internal standard for alcohols andacetone); 5, acetone 13.6 mmoVL; 6, ethylene glycol 17.9 mmoVL; 7,propylene glycol 13.6 mmol/L; 8, 2,3-butanediol 11.0 mmoVL; 9, 1 2-butane-diol 17.9 mmoVL (internal standard for diols). (B)Serum from patient positivefor methanol (14.1 mmol/L) and ethylene glycol (3.0 mmoVL). (C)Serum frompatient positive for ethanol (30.1 mmol/L), isopropanol (4.3 mmoVL), andacetone (17.8 mmolIL).

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Table 1. Retention times of alcohols and diols.Retention time, mm

Table 3. Recovery of alcohols and diols fromsupplemented plasma.

Conc mmoVL

CompoundRtx-200 wide-bore

capillaryHayeSep R

packed column Compound Added Measured % RecoveryMethanol 0.59 2.2 Methanol 49.40 45.21 91.52Ethanol 0.72 4.6 9.88 9.58 96.96Isopropanol 0.85 6.8 Ethanol 34.26 32.09 93.67n-Propanol 1.09 8.0 6.85 6.76 98.69Acetone 1.32 6.0 2-Propanol 26.12 24.34 93.19Propionic acid 1.82 5.22 5.15 98.66Ethylene glycol 2.01 11.2 Ethylene glycol 35.86 35.90 100.11,2-Propanediol 2.17 7.17 7.34 102.42,3-Butanediol 2.35 2,3-Butanediol 22.08 21.52 97.461,2-Butanediol 2.51 4.42 4.46 100.9

Discussion

Traditionally, most laboratories have used separatechromatographic conditions for analyzing alcohols and

302 CLINICAL CHEMISTRY, Vol. 41, No. 2, 1995

the Rtx-200 column and 1 mmol/L on the HayeSep Rcolumn. Day-to-day precision (CV) was as follows: Forthe HayeSep R (n = 52), methanol = 8.9% at 26.51mmol/L and ethylene glycol = 13.6% at 19.6 mmol/L;for the Rtx-200 (n = 30), methanol = 6.4% at 9.8mmol/L, ethanol = 5.0% at 6.9 mmol/L, 2-propanol =

4.7% at 5.2 mmol/L, and ethylene glycol = 6.2% at 6.8mmol/L. The HayeSep R system was linear (r >0.99with deproteiized serum samples) for concentrationsof methanol, ethanol, 2-propanol, and ethylene glycolover the ranges 0-40 mmol/L. Results of least-squaresregression for the Rtx-200 system are summarized inTable 2 (note: linearity is dependent on FID responseand will vary with instrumentation). Recovery studieswere conducted by standard addition technique; re-sults for the capillary method are shown in Table 3.Table 4 lists the therapeutic drugs and metabolitestested and found not to interfere with the wide-borecapillary method. Table 5 lists retention times on thewide-bore system for various other organic compounds.None of these compounds (except for acetaldehyde; seeDiscussion) coeluted with the target analytes.

Compared with the performance of the packed-col-umn method, the wide-bore capillary column providedroughly 10-fold lower detection limits for alcohols anddiols and required less than half the analysis time.Within-run and between-day CVs were also lower by

the capillary GC method.

Table 2. Least-squares statistics for wide-borecapillary system.

Concrange,

Analyte mmol/L Slope r2 Intercept

Methanol 0-50 1.01 0.9995 0.02Ethanol 0-35 1.01 0.9998 0.07Ethylene glycol 0-35 1.00 0.9997 -0.31Isopropanol 0-27 1.00 0.9999 0.00Acetone 0-27 0.87 0.9907 0.06

Table 4. Compounds that do not interfere with alcoholor diol analysis at the stated concentrations.

Compound

AcetaminophenArnitriptylineCarbamazepineDesipramineDigoxinGentarnicinN-Acetylprocain-

amidePhenobarbital

Conc Compound Conc

795 moVL Phenytoin 99 moVL835 nmoVL Primidone 53 &rnoVL60 moVL Procainamide 53 ,rnoVL791 nmoVL Propanofol 855 jmoVL3.8 nrnol/L Quinidine 20 moVL16.1 Lm0VL Salicylate 3.1 mrnoVL34 moVL Theophylline 164 rnoVL

207 1moVL TrobamycinValproic acidVancomycin

16.1 moVL891 1Lm0VL39 moVL

diols. To avoid the system contamination seen withdirect injection of whole blood (12) or deproteiizedserum (4), researchers have used headspace GC foranalysis of volatile alcohols (13, 14). Less-volatile com-pounds such as ethylene glycol usually require derivat-ization (1, 5, 6) before analysis. Aarstad et al. (15)quantitated ethylene glycol without derivatization byusing direct injection onto a packed column; however,their high initial oven temperature did not allow quan-titation of low-boiling alcohols. For laboratories receiv-ing specimens that might contain alcohols and diols, a

two-system approach is too time-consuming and labor-intensive. We therefore developed a single packed-column chromatographic system capable of analyzingalcohols and diols without sample derivatization.

The packed-column method we developed (16) for theanalysis of serum alcohols and ethylene glycol uses amid-polarity, acid-resistant porous polymer packing.The HayeSep R column gave good separation of low-boiling alcohols, was fairly robust, and involved rela-tively simple sample preparation. However, declining

SE, accuracy at medical decision thresholds (e.g., -2mmol/L mmol/L for ethylene glycol) and excessive run times0.562 warranted development of a more rapid and sensitive0.265 assay. Therefore, we decided to update the method to a0.318 wide-bore capillary system.0.149 Several authors have described capillary GC meth-0.318 ods for the analysis of alcohols or ethylene glycol

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0.530.710.87

1.16

1.32

1.45

1.611.731.82

2.062.182.29

2.39

2.852.963.08

3.183.90

Not seen

CLINICAL CHEMISTRY, Vol. 41, No. 2, 1995 303

Table 5. Retention times of various organic compounds on wide-bore capillary system.Compound Retention time, mm Compound Retention time, mm

Monohydroxyalcohols Ketones, aldehydes, etc.Methanol 0.59 FormaldehydeEthanol 0.72 Chloroform

2-Propanol 0.85 DichloromethaneI -Propanol 1.09 ChloralIsobutyl alcohol 1.42 2-Propanone (acetone)tert-Pentanol 1.52 Acetonitrile1-Butanol 1.61 Ethyl acetate3-Pentanol 1.74 2-Butanone3-Methyl-i -pentyn-3-ol 1.75 Propionic acidIsopentyl alcohol 1.93 3-Pentanone1-Pentanol 2.05 Hydroxyacetone2,2,2-Trichloroethanol 2.43 Isopropylacetone4-Methyl-5-thiazole-ethanol Not seen 2,4-PentanedioneDihydroxyalcoho!s Ethchlorvynol1,2-Ethanediol (ethylene glycol) 2.01 Cyclohexanone1 ,2-Propanediol 2.17 Glycerol2,3-Butanediol 2.35 Dimethyl sulfoxide

1,2-Butanediol 2.51 Triethylene giycol1,3-Propanediol 2.58 Oxalic acid1,3-Butanediol 2.652-Methyl-2,4-pentanediol 2.87

1,4-Butanediol 2.91

(2, 5, 17-19). However, we were unable to find any

reports of a single system for analyzing both alcoholsand diols. Despite the fact that a mega-bore column isrecommended as the first step in converting a packed-column method to a capillary system (20), we encoun-tered several problems in transferring our packed-column method to a wide-bore column. These includedcolumn stationary-phase selection, sample prepara-tion, choices of injection mode and technique, injectionport septa, and type of syringe and needle style used forinjection.

Column selection. A nonpolar phase such as dimeth-yl-polysiloxane (used in e.g., DB-1, HP-i, Rtx-1) may beappropriate for analysis of polar compounds (e.g., alco-hols and dials) in nonpolar solvents, as is commonlydepicted in column manufacturers’ literature. How-ever, a 0.53-mm (i.d.) column of reasonable length (30m) cannot separate these salutes when they are in-jected in water or a water-based matrix such as serumor plasma. At the low (30-40#{176}C)initial oven tempera-ture required for analysis of alcohols, the diols remainin the water bolus as it moves down the column. Theresult is extensive peak broadening and flattening,sometimes so severe that it is impossible to distinguishfrom baseline an ethylene glycol peak in fortified sam-ples known to contain concentrations as great as 5mmoIIL. Increasing the initial oven temperature to

125#{176}C(21) facilitates cold-trapping of ethylene glycolwhile allowing the water vapor to proceed down the

column. However, with injection temperatures 25#{176}Cabove the solvent’s boiling point, there is no solvent-effect focusing of peaks, which is necessary for baselineresolution of low-boiling alcohol. Also, acetone coelutes

with isopropanol. Similar results were obtained with a

more polar DB-624 column (6% cyanopropylphenyl-/94% dimethyl-polysiloxane). Even with a retention gapinstalled to facilitate concentration of the alcohols,neither column was suitable for separating aqueousalcohols and dials in a single run.

Highly polar polyethylene glycol (PEG) phases (suchas DB-Wax, HP-20M, Stabilwax) have been used toanalyze alcohols or dials (3, 13). However, we did notattempt our analysis on a PEG column for two reasons:First, the phase cannot separate ethanol from isopro-panol in short, wide-bore columns; second, PEG phasesare more susceptible to damage induced by water,oxygen, and the higher temperatures required to elutesome of the less-volatile constituents of serum. Thistranslates into increased column bleed, with its atten-dant decrease in sensitivity, and shorter column life-time.

Of many phases of intermediate polarity that mightprovide the desired separation, we chase the Rtx-200column for the unique selectivity towards lone pairelectrons provided by its trifluoropropylmethyl-polysi-loxane phase. The highly electronegative fluorine at-oms have a strong affinity for the hydroxyl groups inalcohols and diols, for ketones, and for chlorofluorocar-bons. The elution order of the alcohols may be predictedby boiling point, and by the number of hydroxyl groupspresent. Ketones elute later than their boiling pointwould predict (Table 1), so acetone is more stronglyretained than the group of monohydroxyl alcohols thatare of interest here. This selective retention providesadditional flexibility in terms of usable temperaturesat the beginning of the chromatographic run, therebeing fewer weakly retained compounds to be resolved.The only compounds found to coelute were acetalde-

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304 CLINICAL CHEMISTRY, Vol. 41, No. 2, 1995

hyde and ethanol; however, because serum acetalde-hyde concentrations do not reach mmol/L levels, thisproblem was not judged to be clinically significant.

Sample cleanup. Acid precipitation is a commonlyused method for deproteinizing serum samples. How-ever, bonded-phase columns may be ruined by expo-sure to even small amounts of acidic solutions. To avoidprecipitation of samples with large amounts of salts(which are deposited in the chromatographic inlet, andserve as active adsorption sites), we tested filters ofvarious molecular mass cutoffs from several manufac-turers. All but the UFC3-LGC type (Millipore) useglycerol as a membrane preservative, which is elutedas a very large peak (- 100-fold the internal standardpeak area) late in the chromatographic run. The LCCunits use triethylene glycol, which, while eluting laterthan glycerol, is leached into the sample in amountsusually less than the concentrations of the internalstandards (25 mmol/L). The 10 000-Da cutoff mem-brane removes >99.5% of albumin and >95% of cyto-chrome c (mass -13 000 Da). The flow rate through themembrane is high enough to allow sufficient recovery(-50 p.L) of filtrate after 5 mm in a laboratory micro-centrifuge. The regenerated cellulose membrane can beused with most organic solvents, including acetone, atconcentrations likely to be found in serum. We havevalidated this method for use with serum or plasma;however, whole-blood analysis may be possible withalternative sample preparation.

Internal standards. n-Propanol is widely used as an

internal standard for GC analysis of methanol(4, 14, 22, 23). Packed-column/derivatization methodsfor ethylene glycol have used various internal stan-dards, including 1-3-propanediol (7) and 1,2-pro-panediol (1); recent wide-bore capillary techniques (19)have used 2,3-butanediol. We have found 2,3-butane-diol to be an occasional component of serum samplesanalyzed in our laboratory, and Porter et al. (24)recently suggested that this compound may be a me-tabolite of 2-butanone, a denaturant in alcohol prepa-rations. We elected to use a dual internal standardapproach: n-propanol for alcohols, and 1,2-butanediolfor diols, neither compound being a natural componentof serum. We have also found 1,3-butanediol to be anacceptable internal standard for ethylene glycol.

Injection mode. The injection mode was limited bythe hardware available: Our GC is equipped with anunpurged, packed-column injection port. Instead ofmaking an expensive modification such as installing adedicated on-column capillary port, or a split-injectionport, we obtained capillary-column adaptors and injec-tion-port liners of several types from two manufactur-ers. The best results were obtained by using a 5-mm(o.d.) Uniliner inside a metal adaptor (to reduce thelikelihood of crushing the glass liner while making agas-tight seal).

Direct injection, in which near-instantaneous vapor-ization of injected samples is the goal, is suitable for alarge proportion of GC analyses. After vaporization ofthe sample in an expansion chamber within the port

liner, the analytes of interest are moved to the columnhead by the carrier gas. Nonvolatile, high-boiling com-ponents of the sample are deposited in the port liner.These deposits eventually accumulate in sufficientquantities to affect subsequent analyses, but cleaning(with methanol:dichloromethane:water, 3:1:1 by vol)and deactivation (with dimethyldichlorosilane) of theliners is an accepted part of regular maintenance.

Careful sample cleanup will extend the scheduledmaintenance interval. Ultrafiltration of the serumsamples removes most of the nonvolatile residues,including proteins >10 000 Da, which might otherwisefoul the column. Any contaminants that do accumulatemay be removed by cutting 1 m from the head of theguard column (retention gap). If this does not restoreacceptable chromatography, the capillary column maybe further cleaned by rinsing with methanol:di-choloromethane:water (3:1:1, by vol). In our laboratory,solvent rinsing has not been required mare than onceevery 1000 injections.

Hot on-column injection was attempted to avoid bothdiscrimination and flashback problems. This mode ismade possible by inverting the Uniliner to allow inser-tian of a 26-gauge needle directly into the head af thecolumn. On-column injection yielded well-formedpeaks with no tailing evident as long as the columnhead was clean. However, as nonvolatile sample resi-dues accumulated, active solutes such as alcohols anddials, including ethylene glycol, begin to adsorb to thecolumn, again resulting in underestimation of thesample concentrations of these analytes. Chromato-graphic deterioration was significantly more rapid withthis method than with direct injection.

Choice of septum. The choice of injection-port sep-tum is important with both an-column and directvaporizing modes of injection. Because septum parti-cles deposited directly into the column or trapped in avaporizing chamber act as adsarption sites for alcoholsand diols, a septum resistant to caring will reducesystem maintenance requirements. We found theThermolite septa to exhibit only minimal coring, evenafter -100 manual injections. Further, these septaproduced very low “bleed” signals at high columntemperatures. This contributes to better quantitationof high-boiling solutes, which may coelute with some ofthe bleed peaks observed with other septa.

Needle. Septum caring was further reduced by usinga tapered 26/23-gauge needle for automated injections,or, for manual injections, a blunt-tipped needle ratherthan the 22#{176}-beveledneedle usually recommended.Syringes with remavable needles were used to mini-mize replacement costs when needles were bent orbecame plugged from repeated septum penetration.Modification to a septumless injection port (e.g., byinstallation of a Merlin Microseal) is not possible withour instrument, but for those whose inlet canfigurationis compatible, this option might further prolong theintervals between maintenance.

Syringe. The choice of syringe for injections alsogreatly affected assay reproducibility. For manual in-

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CLINICAL CHEMISTRY, Vol. 41, No. 2, 1995 305

jections with a standard microliter syringe, injectingwater after the sample injection revealed that repeatedsyringe rinsing (>25X) was usually insufficient toremove ethylene glycol and glycerol or triethyleneglycol from the syringe. To avoid this carryover ofviscous salutes between injections, we used a gas-tight(Hamilton 180 1RN) syringe. Without the sealing actionof a Teflon tip on the plunger, septum penetrationallows venting of carrier gas, so that sample is forcedaround the plunger and up the syringe barrel by thecolumn headpressure. In contrast, the rapid plungeraction of the autoinjector sometimes failed to aspiratesample when used with a gas-tight syringe. We there-fore used a standard syringe for the automated injec-tions. The “blowback” seen in manual injections wasnot a problem when we used the injector’s “fast” mode,and no carryover was seen between injects.

In summary, we have developed a rapid, sensitivecapillary GC technique for simultaneous quantitationof alcohols and dials in serum. Several recent papers(25-29) have suggested that low concentrations ofmethanol may be found in alcohol abusers as well as inoccupationally exposed workers and may be a usefulmarker of alcohol abuse (29). The recent “St. Louis”case of misidentification of propionic acid as ethyleneglycol in a patient with methylmalonic acidemia (10)

emphasizes the advantages of our wide-bore system.Our method offers significant improvements in speci-ficity, sensitivity, and turnaround time over existingtechniques. In experienced hands, a sample, calibrator,and quality-control material can be analyzed and re-sults reported in <40 mm.

References1. Balikova M, Kohlicek J. Rapid determination of ethyleneglycol at toxic levels in serum and urine. J Chramatogr 1988;434:469-74.2. Shahangian S, Robinson VL, Jennison TA. Formate concen-trations in a case of methanol ingestion. Cliii Chem 1984;30:1413-4.3. Jonssan JA, Eklund A, Maim L. Determination of ethyleneglycol in postmortem blood by capillary gas chromatography. JAnal Toxicol 1989;13:25-6.4. Cheung S-T, Lin W-N. Simultaneous determination of meth-anol, ethanol, acetone, isopropanal and ethylene glycol in plasmaby gas chromatography. J Chromatagr 1987;414:248-50.5. Smith NB. Determination of serum ethylene glycol by capil-lary gas chromatography. Clin Chim Acta 1984;144:269-72.6. Flanagan RJ, Dawling S, Buckley BM. Measurement of ethyl-ene glycol (ethan-1,2-diol) in biological specimens using deriva.tisation and gas-liquid chromatography with flame iomsationdetection. Ann Clin Biochem 1987;24:80-4.7. Doedens DJ. Methods for the determination of ethylene glycol.Vet Hum Toxicol 1983;25:96-101.8. Hansson P, Masson P. Simple enzymatic screening assay forethylene glycol (ethane-1,2-diol) in serum. Clin Chim Acta 1989;182:95-102.

9. Vinet B. An enzymatic assay for the specific determination ofmethanol in serum. Cliii Chem 1987;33:2204-8.10. Shoemaker JD, Lynch RE, Hoffman JW, Sly WS. Misidenti-fication of propionic acid as ethylene glycol in a patient withmethylmalonic acidemia. J Pediatr 1992;120:417-21.11. Jones AW, Nilsson L, Gladh SA, Karsson K, Beck-Friis J.2,3-Butanediol in plasma from an alcoholic mistakenly identifiedas ethylene glycol by gas-chromatographic analysis. Chin Chem1991;37:1453-5.12. Jacobsen D, Jansen H, Larsen-Wiik E, Bredesen JE, Hal-vorsen S. Studies on methanol poisoning. Acta Med Scand 1992;212:5-10.13. Brown DJ, Long WC. Quality control in blood alcohol analy-sis: simultaneous quantitation and confirmation. J Anal Toxicol1988;12:279-83.14. Fraser AD, MacNeil W. Gas chromatographic analysis ofmethyl formate and application in methanol poisoning cases. JAnal Toxicol 1989;13:73-6.15. Aarstad K, Dale 0, Aakeruik 0, Ourebo 5, Zahlsen K A rapidgas chromatographic method for determination of ethylene glycolin serum and urine. J Anal Toxicol 1993;17:218-21.16. Perkins SL, Maddock M, Livesey J. Simultaneous gas chro-matographic analysis of plasma alcohols and ethylene glycol. ClinBiochem Rev 1993;14:295.17. Danielson JW, Snell RP, Oxborrow GS. Detection and quan-titation of ethylene oxide, 2-chloroethanol, and ethylene glycolwith capillary gas chromatography. J Chromatogr Sci 1990;28:97-101.18. Boneva S. Wide-bore capillary column for the direct analysisof ethanolamines and ethylene glycols. J Chromatogr 1991;31:171-2.19. Edinboro LE, Nanco CR, Soghioan DM, Poklis A. Determina-tion of ethylene glycol in serum utilizing direct injection on awide-bore capillary column. Ther Drug Monit 1993;15:220-3.20. Jennings W. Instrument conversion and adaptation. In: Jen-nings W, ed. Analytical gas chromatography. San Diego: Aca-demic Press, 1987:153-63.21. Schmid RW, Wolf CH. Large volume sample application by asimplified “closed” on-column injector in capillary gas chromatog-raphy. J Chromatogr 1989;27:221-4.22. Pla A, Hernandez AF, Gil F, Garcia-Alonso M, Villanueva B.A fatal case of oral ingestion of methanol. Distribution in post-mortem tissues and fluids including pericardial fluid and vitreoushumor. Forensic Sci mt 1991;49:193-6.23. Caldwell K Methanol levels in methylated spirit drinkingalcoholics. N Z Med J 1986;99:764-5.24. Porter WH, Jarrells MC, Sun DH. Improved specificity forethylene glycol determined as the phenylboronate by capillarycolumn gas chromatography. Cliii Chem 1994;40:850-1.25. Kawai T, Yasugi T, Kazunori M, Horiguchi S, Hirase Y,Uchida Y, Ikeda M. Methanol in urine as a biological indicator ofoccupational exposure to methanol vapor. mt Arch Occup Envi-ron Health 1991;63:311-8.26. Yasugi T, Kawai T, Mizunuma K, Horiguchi 5, Iwami 0,Iguchi H, Ikeda M. Formic acid excretion in comparison withmethanol excretion in urine of workers occupationally exposed tomethanol. hit Arch Occup Environ Health 1992;64:329-37.27. Schuberth J. Volatile compounds detected in blood of drunkdrivers by headspace/capillary gas chromatography/ian trap massspectrometry. Biol Mass Spectrom 1991;20:699-702.28. Jones AW, Lowinger H. Relationship between the concentra-tion of ethanol and methanol in blood samples from Swedishdrinking drivers. Forensic Sci mt 1988;37:277-85.29. Rome RP, Eriksson CJP, Yhikahri R, Penttila A, SalaspuroM. Methanol as a marker of alcohol abuse. Alcohol Clin Exp Res1989;13:172-5.


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