Disposable reversed phase chromatography columns for improved detection of carboxylic acids in body fluids by electron capture gas liquid chromatography

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JOURNAL OFCLINICALMICROBIOLOGY,July 1987,p. 1216-1220 0095-1137/87/071216-05$02.00/0

Disposable

Reversed-Phase

Chromatography

Columns for

Improved

Detection of

Carboxylic

Acids

in

Body

Fluids

by

Electron-Capture

Gas-Liquid

Chromatography

M. I. DANESHVAR,* J. B. BROOKS, ANDR. M. WINSTEAD

AnalyticalChemistry Laboratory, Meningitis andSpecialPathogens Branch, DivisionofBacterialDiseases, Centersfor Disease Control, Atlanta, Georgia 30333

Received21 November1986/Accepted6April 1987

Disposable reversed-phase chromatography columns were tested for their effectiveness in removing unreacted trichloroethanol (TCE) from derivatized samples for gas-liquid chromatography analysis.

Derivatized acidicchloroformextractsofsaponified whole cellsofMycobacterium species, spentculturemedia, andderivatized acidic chloroformextractsofserumand cerebrospinal fluids frompatientswith tuberculous meningitisweretested.Sampleswereaddedtopreconditionedreversed-phasechromatography columns, and various solvents and solvent mixtures were tested to determine maximumrecovery of the TCEderivatives. With thisprocedure, we wereabletoquicklyremovetheTCEreagentandefficientlyrecoverTCE-derivatized carboxylic acids. Use ofthese columnsimprovedthereagentcleanup procedure, simplifiedthederivatization step, permitted increased detection of tracecomponents, such astuberculostearic acid, in body fluids, and improved theselectivity of the procedurefordetectionofcarboxylic acids.

One problem associated with derivatization of sample

extracts for analysis by frequency-pulsed electron-capture gas-liquid chromatography (FPEC-GLC) is the removal of excesselectron-capturingreagent. Failuretoremove excess

reagentlimitstheamountofsample thatcanbeanalyzed and

thusreducesdetection of important metabolites thatmaybe present in body fluids at femtomole (10-') or picomole (10-12) quantities. Disposable reversed-phase

chromatogra-phy (RPC) columnsweretested for theireffectivenessinthe removal of unreacted trichloroethanol (TCE) from deriva-tized samples. The basis for reversed-phase sorbent

extrac-tion is that the solid phase has agreater attraction for the

TCE-derivatized acid than for the solvent in which the derivative isdissolved (5). The RPC column makesuse ofa

solidphase of silicatowhich is bondedpacking material with

hydrophobic functionality. Usually the RPC column material hasa2-, 8-,or18-hydrocarbonchain functionalgroupwhich is bonded to the solid silica. If it is necessary to remove

unreacted TCE from the derivatized sample, an RPC

col-umn-packing material is chosen that is nonpolar, and the polar TCE reagent is bound to the column by hydrophilic

interaction. The less polar acid TCEestersaresubsequently

eluted withasolvent of low polarity. Elutioncanbe

accom-plished by choosing an appropriate nonpolar solvent (hexane)orbyacombination of solventssuch as

methanol-chloroform (MeOH-CHCl3) which selectively elute short-chain,morepolar TCEesters (C2toC6)orlong-chain, less

polar TCE esters (C7 toC22). Thepurpose ofthis

investi-gation was to determinethe effectiveness, practicality, and

time-saving features of RPC columns for the removal of unreacted TCE reagent. Afurther goal was to simplify the

derivatization procedure previously described (1) by elim-inating certain time-consuming steps.

* Corresponding author.

MATERIALS ANDMETHODS

Body fluids, spent culture media, or saponified cellular material (2 ml) was adjusted to pH 2 with 0.1 ml of50%

(vol/vol) sulfuric acid-distilled water. Internal standards

were added (1), and the sample was extracted with nanogradechloroform (CHCl3;Mallinckrodt, Inc.)toobtain

carboxylic acids as previously described (1-3). The acidic CHCl3 extracts were then derivatized with TCE to form

highly electron-absorbingTCE esters of the carboxylic

ac-ids. The TCE derivatives were prepared by one of the following procedures.

Procedure 1. The acidicCHCl3extract (20ml)was

evap-orated withcleandry airto25 ,ulaspreviously described(1). Then, 25 ,ul of a freshly prepared mixture (1:9) of

TCE-CHCl3 was added to the sample. Next, 30 ,ul of

heptafluorobutyricanhydridewasaddedtocatalyze the TCE

esterification,andthereactionwaspermittedtostandfor 30 min. CHCl3 (200 ,ul)wasadded, and the derivatizedsample was acid and base washed to remove excess heptafluoro-butyric anhydrideaspreviously described (1).Next, 100 ,ul ofxylene was added, and the samplewas transferred toa 3-ml conical test tube (Kimble, Div. Owens-Illinois) and evaporated in a 100°C sand bath with clean dry airto the 10-pli mark. Then, 100 pi of xylene-ethanol (50:50) was addedasthe final solvent forFPEC-GLC analysis.

Procedure 2. The TCE derivative was prepared as

de-scribed for procedure 1 above by adding TCE and

heptafluorobutyric anhydride reagents to the concentrated acidicCHCl3extract,and the samplewaspermittedtostand for30 min. Next, 200 pil of CHC13 and 100pi of xylenewere

added to the unwashed derivatized sample and evaporated withcleandry air ina100°C sand bathtoabout25 pul.Then, 400 ,ul ofnanograde hexane (Mallinckrodt) was added as

solvent forcleanupwith an RPC C18 column (Analytichem International, Inc.). The C18 column was conditioned by

placing it onto a VAC-ELUT vacuum manifold cover

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w C0qr- o c

cri C O

0 0

w

CQ w

Q~~~~~~~

w

a.) - C m M W

C.',~~~~~~~~~~~~~~~~~~~~~~~~C

C,~~~~~~~~~~~v

2 30 4b eN i0O71

10

40/inTm

5020

70C

FIG. 1. FPEC-GLCchromatogramsof TCE-derivatizedsamples

from standard acid mixtures C2toC22. TCE derivativesareshown aftercleanup withaC2 RPC column and various eluting solvents and a solvent mixture. Analyses were done on a 25-m OV-225

capillary column. The letter C followed by a number indicates a

saturatedcarboxylicacid with the number of carbonatomsindicated

bythenumber. A,TCE derivativepassed throughC2RPC column eluted with methanol(complete elution);B,TCEderivativepassed throughC2 RPC column eluted with chloroform; C,TCEderivative

passed through C2 RPC column eluted with methanol-chloroform

(30:70, vol/vol).

(Analytichem) and aspirating 6 ml of MeOH through the

column. The MeOH level was maintained at thetop of the

sorbent,and 6 mlof hexanewasadded andaspirated through

the column. The level of hexane was held at the topof the

sorbent,and thederivatizedsample,inhexane,wasaddedto

the column. Next, a partial vacuum of about 2 inches (ca.

6.77kPa) ofHgwasappliedtothecolumn, and hexanewas

aspirated until the sorbent was dry (about 2 min). The

solvent wasdiscarded, andacollection tube (10by 75mm;

Corning Glass Works) was placed on the collector rack

inside the VAC-ELUT vacuum manifold. The TCE esters

werethen elutedfrom the RPC column with 3 ml of hexane.

Thesamplewas evaporatedtoabout 100 pilina 100'Csand

bath,and 100 ofxylenewasadded.Next,thesamplewas

transferred to a 3-ml conical test

tube,

placed

in the 100'C sand

bath,

and

evaporated

with clean

dry

air to the

10-pil

mark,

and 100

ktl

of

xylene-ethanol (50:50)

wasaddedasthe final solvent for FPEC-GLC

analysis.

Derivatives

prepared

as described for

procedure

i were

further tested for additional TCE

cleanup

and removal of short-chain acids with RPC columns as follows.

C18, C8,

and C2 RPC columns were conditioned with 2 column volumes ofMeOH followed

by

2column volumes of acidi-fied water.

Then,

the TCE-derivatized

sample

dissolved in 100

ktl

of

xylene-ethanol (50:50)

was added to the

column,

and the aqueous

xylene-ethanol

was

aspirated

through

the column. The

sample

wasthen elutedfrom the columns with variousratiosof

CHC13-MeOH

ranging

from 70:30to80:20.

Finally,

the

sample

was

evaporated

to 25

pi1,

and 100 pilof

xylene-ethanol

was added. The TCE esters were

analyzed

on a Perkin-Elmer 900 or 3920 gas

chromatograph.

The instrumentswere

equipped

withdual FPEC-GLC detectors. Two

large-bore nonpolar

fused-silica

bonded-phase

capillary

columns,

coated with a

4.4-ktm-thick

film of

0V-lOi,

were

used. One of the 0V-lOi columns was 10 m, and the other column was 25 m

long.

Samples

were also

analyzed

with a

polar

fused-silica

bonded-phase capillary

column

(0.32

mm

[inside

diameter]

by

25

m)

coatedwith a0.25-ptm-thick film of OV-225. Helium was usedas the carrier gas in allof the

capillary

columnsatafiowrateof S ml/min. The

makeup

gas for each

capillary

column was a mixture of95% argon and

5%methane. Thecombined fiow rate of the carrier gas and the

makeup

gas

through

the 63Ni FPEC detector was 70

ml/min. For

analysis

of the TCE-derivatized acids on the 10-m0V-lOi

column,

theinstrumentwasheldisothermal at 90'C for 3 min and then

programmed

to 2750C at a linear increase of 60C/min. For the 25-m 0V-lOi column, the instrument washeldat90'C for 2 min and then

programmed

at 40C/min to 2750C. For the

polar

OV-225

column,

the

instrumentwasheldat100'Cfor 3 min and then

programmed

to 220'C at a linear increase of 20C/min. The derivatized

sample

(1

pI)

was

injected

ontothecolumnsfor

analyses.

An IBM

System

9000computer

equipped

with CAP 1.4software

(IBM

Corp.)

was used to

integrate

the

peaks, expand

sec-tions of the

chromatograms

for easy

comparison,

and eval-uate various levels ofattenuation.

RESULTS

TCE-derivatized standard mixtures of

carboxylic

acids

(C2

to

C22),

along

with derivatized

body

fiuid and spent

culture medium

samples

that had been derivatized and

processed

as described for

procedure

1,

were tested for further removal ofexcess TCE with

C2, C8,

and C18 RPC columns. In

addition,

various solvents and solvent mixtures

were tested to determine the best recovery of the acids. Polar solvents, such as

MeOH,

eluted both the derivatized acids and the excess TCE reagent

(Fig. lA). Moderately

polar solvents,

such as

CHC13,

didnotelutetheexcessTCE

reagent and also reduced the recoveryof shorter-chain acid

derivatives

(C2

to

C1O)

on the C2 RPC column

(Fig. lB).

Loss of these short-chain acids

might

be desirable if the

major

purposewereto testfor

long-chain (ClO

toC20) acids,

which are found in whole bacterial celis. Selective elution

was tested forrecovery of the shorter-chain acids

by

com-bining MeOH-CHC13

inratios of 30:70

(vol/vol)

(Fig. 1C)

or

25:75 to

produce

the desired

polarity.

MeOH-CHC13

at a

ratio of 25:75

proved

to be the best

eluting

solvent with

samples

derivatized as in

procedure

1, because the 30:70 mixture often eluted many of the TCE-derivatized

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1218 DANESHVAR ET AL.

chain(C2 toC7) acids. For further studiesof acidrecoveries, samples were added to various preconditionedRPCcolumns with C18, C8, and C2 functional groups by using MeOH-CHC13 (25:75) as the eluting solvent. The following results

were obtained. Unreacted TCE (Fig. 2A) and derivatized acids withchainlengths up to C10were retainedbytheC18 RPC column, whereas acids with chain lengths longer than C10 were released (Fig. 2B). The C8 RPC column retained unreacted TCE and derivatized acidswith chainlengths up toC8 and released acids with chain lengths longerthan C8

(Fig.2C).TheC2RPC columnretained unreacted TCEand derivatized acids with chain lengths up to C6 and released acids with chain lengthslongerthan C6 (Fig. 2D). Because the C2 RPC column retained the unreacted TCE and

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FIG. 3. FPEC-GLCchromatograms of TCE-derivatizedsamples from standard acid mixtures C2to C22. Thederivatives were not

acid andbase washed butwerecleaned upby C18 RPC columns. Analyses were done on a 25-m OV-101 capillary column. For definitions of abbreviations, see the legend to Fig. 1. A, TCE derivative passed through C18 RPC column without precolumn xylene-airevaporationcleanup;B,TCEderivativepassedthrough C18 RPC column without postcolumn xylene-air evaporation cleanup; C, TCE derivative passedthroughC18 RPC column with pre-andpostcolumn xylene-airevaporationcleanup.

-i

LD

0 5 10 15 20 25 30 35 40

Time

90 C 60 C/Min 2750C

FIG. 2. FPEC-GLCchromatogramsofTCE-derivatized samples from standard acid mixtures C2 to C22. Derivatives are shown

before (A) andafter (B, C, andD)various RPCcolumn cleanups withMeOH-CHCl3(25:75[vol/vol])astheeluting solvent. Analyses

were-done on a 10-m OV-101 large-bore capillary column. For

definitions of abbreviations, see the legend to Fig. 1. A, TCE

derivative, cleanup byxylene-air evaporation; B, TCE derivativeas

inchromatogram A, passed through C18 RPC column, sample in

xylene-ethanol(50:50), eluentCHC13-MeOH(75:25);C,TCE deriv-ativeasinchromatogramBexceptpassedthrough C8 RPC column;

D, TCEderivativeasin chromatogramCexceptpassedthrough C2 RPCcolumn.

leased more of the shorter-chain acids, it was chosen for routine use to clean up derivatized samples of body fluids which had been derivatized previously by procedure 1. Once the cleanup of these samples was accomplished, webegan

research in theuseofRPCcolumns to shorten the derivati-zation steps described for procedure 1. We foundthat the solventsystemdescribed forprocedure 2 gave theminimum

loss of the TCE esters and removed the most unreacted

TCE. Figure 3A shows the results obtained under the best solventconditions with noxylene-air evaporation before the TCE-derivatized sample was added to the RPC column. Observe that there isanoticeable tailing and overloadingof

the columns from the excess (unreacted)TCE. Theresults obtained when the xylene-airevaporationwasomittedafter the sample had been eluted from the column are shown in Fig. 3B. Figure 3C shows theresults obtainedwhen

xylene

-J.CLIN. MICROBIOL.

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bepositivefor tuberculous meningitis byapositivepurified

proteinderivative skintestand response to drug therapy. DISCUSSION

Theuse ofelectron-absorbingreagents such as

heptafluo-robutyric anhydrideandTCE, although essential for specific functionalgroupderivatizationtoproduce highlydetectable

electron-capturing derivatives, presentsproblems when

an-alyzedby FPECdetectors and capillary columns.The useof TCEestersoffersaneffectivemeansfordetectionof metab-A olitesin diseased body fluids (3, 4), but effective removal of these reagents is essential to prevent overloading of the FPEC detector and capillary columns. Removal of the excess TCE reagent without significant loss of the TCE esters permits the application of more sample onto the capillarycolumns, which results in increased sensitivity.

Thecomplete removalofunreacted TCE reagentwithout significant loss of the short-chain TCE esters provedto be difficult. We made severalattempts to removeallor mostof theunreacted TCE without prior cleanup by usingC18,C8, C2, CN, and silicon extraction columns. We also tried different solvents and solvent combinations. Whentheonly P

B cleanup procedure used was the extraction column, the

maximum removal ofTCE reagent was obtained by useof

c

10 20 30 40 50 60

90°C 4°C/Min Time 275°C

FIG. 4. FPEC-GLCchromatogramsofTCE-derivatizedsamples from standard acid mixtures C2 to C22 shown to demonstrate

reproducibility.Thederivativeswerenotacidand base washed but

werexylene-air evaporated, cleanedupbyC18RPC columns, and finally cleanedupbyasecond xylene-air evaporation. The

FPEC-GLCconditionswere thesameasthosedescribedinthelegendto

Fig. 3.Fordefinitions ofabbreviations, seethelegendtoFig.1. A, B, andC,TCE derivativepassedthroughC18 RPC columnwith

pre-andpostcolumn xylene-air evaporation cleanup.

air evaporation was done both before and after the sample wasputthrough the RPC column. The xylene-air

evapora-ticn stepwith the sand bath (100°C) and clean dry air was easyto accomplish and reproducible. Figures4A, B, and C show thedegree of reproducibility obtainedinthreeseparate derivatizations with the C18 RPC column and thexylene-air cleanup describedforprocedure 2.Shown inFig. 5AandB

arechromatograms of cerebrospinalfluidfromapatientwith

suspected tuberculous meningitisderivatizedandprocessed throughtheC18 RPC columnas described forprocedure2. Tuberculostearic acid, a known cellular constituent of Mycobacterium tuberculosis (P. A. Mardh, L. Larsson, N.

Hiby, H. C.Engback, and G.Odham, Letter, Lanceti:367, 1983), was present in small amounts and was resolved on

bothcolumns. Subsequently, thepatientwasdeterminedto

ui

cc 0

uJ-r

S

-i

i

i.

t-e

l

UÏ

oCD0

A

10 20 30 40 50 60

900 40C/Min Time 2750

c.> n~~~~~~~~~~~~~~L

c.>~~~~~~~t

10

ci»,

FI

B

20 30 40 50 60

1000C 2°C/Min Time 2200C

FIG. 5. FPEC-GLC chromatograms of TCE-derivatized acidic

chloroformextractofcerebrospinalfluid fromapatientwith tuber-culous meningitis. The cleanup proceçlure was the same as that

described in the legend to Fig. 4. Use of a colon between two numbers indicates unsaturation. For definitions ofabbreviations, see the legend to Fig. 1. A, TCE derivative, C18 RPC cleanup,

CA2841cerebrospinalfluidfromapatientwith tuberculous

menin-gitis, OV-101 capillary column analysis; B, same as in

chromatogramAexceptanalyzedon anOV-225capillary column.

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1220 DANESHVAR ET AL.

the C18 column and hexane asboth the sample solventand the eluent. We also tried passing the sample through two

consecutive columns, but we were unable to completely remove excess TCE reagent. Finally, we had to incorporate a xylene-air evaporation step as described for procedure 2. Two xylene-air evaporations were necessary to obtain the

desiredresults; both werereproducibleand easy toperform. Thefinal xylene-air evaporationofprocedure2 must bedone

carefullytoobtainreproducible results. Preparation of TCE esters of carboxylic acids as described for procedure 2 simplifies the derivatization steps by eliminating the acid and

base washes. Moreover, six to eight derivatives can be

conveniently prepared at the same time.

Although

not totally selective forTCE esters, use of the

C18 RPC column increased the selectivityand sensitivityof the FPEC-GLC analysis of TCE esters of carboxylic acids, and use of the improved derivatization procedure gives a

cleaner analysis (less background) andincreases sensitivity by permittingthe injection of more sample.

J.CLIN. MICROBIOL.

LITERATURE CITED

1. Alley, C. C., J.B.Brooks,and D.S.Kellogg, Jr.1979. Electron capture gas-liquid chromatographic-mass spectral identification ofacidsproduced byNeisseriameningitidis inadefined medium. J.Clin. Microbiol. 9:97-102.

2. Brooks, J. B., C. C. Alley,andJ. A.Liddle. 1974. Simultaneous esterification of carboxylic and hydroxyl groups with alcohol and heptafluorobutyric anhydride for analysis by gas chromatogra-phy.Anal.Chem.46:1930-1934.

3. Brooks, J. B.,D.C. Edman, C. C. Alley,R. B.Craven,andN. I. Girgis.1980.Frequency-pulsed electron capture gas-liquid chro-matography and thetryptophan colortestforrapid diagnosis of tuberculous and other forms oflymphocytic meningitis. J. Clin. Microbiol. 12:208-215.

4. Coonrod, J.D.,L.J. Kunz,and M.J. Ferrace.1983. The direct detection of microorganisms in clinical samples, p. 313-334. Academic Press, Inc.,NewYork.

5. Van Horn, K. C. 1985. Sorbent extraction technology. Analytichem International, Inc., Harbor City, Calif.

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