CLIN. CHEM. 22/6, 739-748 (1976)

CLINICALCHEMISTRY, Vol.22,No. 6.1976 739

Analytical Toxicology: Applications of Element-Selective

Electrolytic Conductivity Detection for Gas Chromatography

Brian E. Pape

We applied a commercially available microelectrolyticconductivity detector to toxicological problems of quali-tative and quantitative analysis by gas chromatography.The detector can be used for the sensitive and selectivedetection of halogen-, nitrogen-, and sulfur-containingcompounds. Relative response in different element-se-lective detector variables such as reaction gas, reactioncatalyst, and furnace temperature can be used to furtherimprove qualitative identification by gas chromatography.

AddftionalKeyphrases: drug assay #{149}flame ionization de-tector #{149}electron capture analysis #{149}detection of N-, S-,and halogen-containing compounds in blood

Gas-liquid chromatography (GLC)1 is a fundamental

analytical tool in most clinical and forensic toxicologylaboratories. Most such instrumentation in these lab-oratories makes use of flame ionization detection (FID)because of its good general response, adequate sensi-tivity for most common analysis, excellent linear rangeand detector stability, and few problems with the in-

strumental interface and operational variables such ascombustion gases, ion collection, and signal amplifica-tion. Analytical requirements, however, continually

arise for which greater detector sensitivity and selec-tivity is needed than FID can supply. Some examples

are halogen-containing volatiles in complex mixture,specificity for sulfur-containing phenothiazines andphenothiazine metabolites, and selectivity for nitro-gen-containing organic compounds as compared withhydrocarbon co-extractables. Improved detector sen-sitivity is most often required in analyses for basic drugsin blood and tissues.

A summary of some common types of detectors

(thermal conductivity, gas density balance, electroncapture, alkali flame, helium ionization, and cross sec-tion) by McNair and Bonelli (1) illustrates the com-

promises that must often be made in increasing detectorsensitivity and (or) selectivity for an element or func-

tional group while retaining the required flexibility

Department of Pathology-Toxicology Laboratory, School ofMedicine, University of Missouri-Columbia, Columbia, Mo. 65201.

1 Nonstandard abbreviations used: GLC, gas-liquid chromatog-raphy (-ic); FID, flame ionization detector; and EICD, electrolyticconductivity detection (-or).

Received Sept. 4, 1975; accepted Feb. 26, 1976.

in GLC instrumentation. Other detector systems usedin GLC include the thermistor, argon, microwave

emission, photoionization, nonradioactive helium ion-ization, radiofrequency, ultrasonic, microcoulometric,

reaction coulometric, electrolytic conductivity, infraredspectrometer, photometric, mass spectrometer, far ul-traviolet, and spectrofluorometric systems. The per-formance characteristics of these have been reviewedby Hartman (2). The flame photometric detector and

electron capture detector have been used to detect

sulfur- and halogen-containing drugs, respectively.Photometric systems are adequately sensitive and quite

selective, but their response is nonlinear and their ap-plication limited. Most applications of electron capturedetection involve the formation of halogenated deriv-atives, requiring additional analytical preparation.Functional-group chemistry of some major drug classesis not favorable to such derivitization reactions (e.g.,

unsubstituted nitrogen heterocyclics and tertiaryamines). As a result of these limitations, other ele-ment-selective detector systems are receiving increased

attention. For example, Toseland et a!. (3) have appliedto thermionic nitrogen-selective detector to the GLCdetermination of anticonvulsants and barbiturates inplasma and tissues.

The electrolytic conductivity detector represents

another element-selective system that as yet has beeninvestigated only casually. The application of electro-

lytic conductivity detection (E1CD) to gas chromatog-raphy was first reported by Piringer and Pascalau (4).Coulson (5) later developed a modified E1CD systemthat allowed the selective and sensitive detection oforganic halogen-, sulfur-, and nitrogen-containingcompounds. Jones and Nickless (6) constructed amodified Coulson detector with a slightly greater sen-sitivity for halogenated compounds. The commercially

available Coulson E1CD system, while quite sensitive,suffered from its large size, its required interfacing, andits lower sensitivity as compared to electron capturedetection of halogenated pesticides. The instrumentalevolution and applications of E1CD have been reviewedby Selucky (7). Hall (8, 9) subsequently reported theenhancement of the sensitivity and selectivity of theCoulson detector and constructed an improved mi-

Fig. 1. Block diagram of the EICD system

7A11(‘I IkIlf’AI (‘UCIAIOTDV !,.I ‘s’) k1.

croelectrolytic conductivity detector, which is nowcommercially available (10).

The principle of E1CD operation. Effluent from a gaschromatograph undergoes thermal decompositionunder predetermined conditions of furnace tempera-ture, reaction gas, reaction catalyst, and abstractors, andis then combined with a stream of de-ionized liquid ina simple gas-liquid contractor. The electrical conduc-tivity of the liquid is continuously measured with an a.c.bridge circuit and auxiliary recorder. Only those com-ponents are detected in the gas chromatographic ef-fluent that are decomposed to yield products that areboth readily soluble and ionized in the conductivityliquid. Figure 1 shows the relationship of the principalcomponents in the E1CD system, all of which are con-

tained in a single unit.The pyrolyzer section (A and B) contains the detector

furnace, the quartz or nickle reaction tube, and the re-action gas inlet. The cell assembly (C and D) consistsof an integrated one-piece Teflon gas-liquid contractorand micro-conductivity cell made of stainless steel andTeflon. The liquid circulation system (E and F) consists

of a glass liquid reservoir, liquid pump, needle valve forflow regulation, and an ion-exchange bed. Componentsof the conductivity bridge include a fixed-voltage a.c.power supply, current-suppression bridge, and signalattenuator. Conductivity-bridge and electrometer-output controls consist of an attenuator, which selectsone of eight binary levels of output signal attenuation;a conductivity range switch, which selects one of sevenranges of conductivity measurement representingfull-scale pen deflection, from 1-1000 tQ; a coarse andfine zero control, which balances the a.c. bridge to allowpositioning of recorder pen; and a display meter se-lectable for furnace temperature or cell conductivity.

Additional components of the detector system includea furnace temperature controller, and furnace power

and detector power monitors.An appreciation of detector variables is critical to an

understanding of E1CD element-selectivity. Thermaldecomposition of the GLC effluent is based on the re-duction (H2 reaction gas), oxidation (02 or air reactiongas), or pyrolysis (no reaction gas) of the organic speciesat high temperature (usually 800 to 900 #{176}C).Decom-

position may involve either catalytic (quartz reactiontube and nickle wire, or nickel reaction tube) or non-

catalytic (quartz reaction tube) conditions. The par-ticular mode of decomposition may be referred to as the

“furnace chemistry.” The reaction products obtaineddepend on the molecular structure, furnace chemistry,

and the use of post-furnace abstractors that trap acidicor basic products. Solubilization and ionization of thereaction product(s) (NH3, HC1, Cl2, SxOx, etc.) at thegas-liquid contactor may be referred to as “cell chem-istry.” Cell chemistry is based on effective gas-liquidcontact and separation, on ionization processes, and theuse of reactive solutes in the liquid phase. Changes inconductivity attributable to specific conductance, liquidflow rate, and electrometer settings are often termed“conductance variables.”

Selectivity and sensitivity are determined by a par-ticular combination of furnace chemistry, cell chemis-try, and conductance variables (8). In the reductionmode, H2S, HC1, NH3, and CH4 are the major reaction

products obtained from organic compounds containingsulfur, chlorine, or nitrogen. H2S has a low ionizationconstant and gives no appreciable response, HC1 can beremoved by a scrubber such as Sr(OH)2 or AgNO3, andthe formation of NH3 often requires the use of nickle

catalyst. In the oxidative mode, SO2 and SO3, HC1, C02,H20, and N2 are produced from the thermal decompo-sition of sulfur-, chlorine-, and nitrogen-containingorganics. The response produced by CO2 is low, owingto poor solubilization (short gas-liquid contact time)or the use of an organic conductivity solvent (usuallyalcohols); water and N2 give little or no response; sulfur

oxides can be removed from the furnace reactionproducts by using a CaO scrubber; and HC1 can also beremoved with an acid scrubber (AgNO3). Consequently,a proper choice of reaction conditions-abstractors-and of conductivity liquid allows the highly selective

detection of either sulfur-, halogen-, or nitrogen-con-taining compounds.

Further selectivity among organochlorine compounds(example: aliphatic as compared with aromatic chlorine)may be realized by choice of furnace temperature (8).

The highly sensitive detection of organic nitrogen withuse of an HC1/ethanol conductivity solvent has also beenrecommended by Hall (9). The neutralization reaction(NH3 + H30 - NH4 + H20) is believed to produce

a greater change in conductance as compared to solu-bilization and partial ionization (NH3 + H20 -‘ NH4+ OW; pKb = 4.76) (6). Moreover, dilute HC1 con-ductivity solutions may be used to differentiate chlo-rine- and nitrogen-containing organics (reductive mode,nickle catalyst) without the use of selective abstractors(6). Differentiation is based on the decrease in con-

ductivity caused by NH3 neutralization reactions, asopposed to the increase caused by HC1. By analogy, theselective enhancement of E1CD response to sulfur (asH2S) in the reductive mode may be realized by using areactive conductivity solvent (6). Increased sensitivityis based on the rapid oxidation of H2S in dilute acidsolution containing iodine (H2S + ‘2 2W + 21 + 5).

We have used the commercial Hall E1CD system (10)

in routine and special toxicological analysis for some

CLINICALCHEMISTRY, Vol.22,No.6,1976 741

nine months and believe it offers significant advantagesin element-selective GLC screening procedures and inquantitation of nanogram amounts of sample.

Selected E1CD applications to analytical toxicologyand a discussion of general detector principles are thesubject of this report.

Materials

Gas Chromatograph and Detectors

GLC analyses were done with a four-column ModelMT 222 gas chromatograph equipped with E1CD, FID,and flame photometric detector systems (TRACOR Inc.,

Austin, Tex. 78721). The conventional E1CD reaction-gas inlet was modified with two nupro metering valvesand a bypass valve to a soap-bubble flowmeter, allowing

convenient switching between H2 and 02 reaction-gassources and measurement of reaction-gas flow. Helium

carrier gas (25 ml!min) was used because of the slightreduction of N2 to NH3 when N2 carrier gas is used in

conjunction with the E1CD in the reductive mode. The1-mm and 4-mm (i.d.) furnace reaction tubes were madeof quartz, and were obtained from TRACOR Inc. Whenusing the 1 mm reaction tubes, and H2 and 02 reac-tion-gas flow rate was 20 ml! mm in the reductive and

oxidative mode, respectively. With the 4 mm reaction

tubes, reaction-gas flow rate was increased to 75 ml/minto avoid peak broadening owing to increased residencetime in the reaction tube. The 4-mm reaction tubes wereonly used for the analysis of 1 ,4-benzodiazepines and1,4-benzodiazepin-2-ones, tricyclic antidepressants, andphenothiazines (discussed below). When nonhydroc-

arbon solvents were injected and 4-mm reaction tubeswere used, the solvent front was vented ahead of theE1CD furnace, to avoid carbonization of the reaction

tube. The E1CD reaction catalyst, when used, consistedof fine nickle wire, tightly coiled and positioned in the

center of the furnace heating zone (quartz reaction-tube). The E1CD conductivity solvent used for allanalyses except 1,4-benzodiazepines and 1,4-benzodi-azepin-2-ones, tricyclic antidepressants, and pheno-thiazines was isopropanol!distilled water (5/95 by vol)at a flow rate of 0.5-1.0 ml/min cell flow, and wasmaintained at pH 6.5-7.0 by a mixed bed ion-exchangeresin (Amberlite IRN-150; Rohm and Haas, Philadel-phia, Pa. 19105; or from TRACOR Inc.). The other

analyses (noted above) were done by using a conduc-tivity solvent of isopropanol/distilled water (15/85 byvol), maintained at an alkaline pH with a coventional

stacked ion-exchange resin tube containing 70%Soxhlet-extracted IRN-78 on the pump side and 30%Soxhlet-extracted IRN-150 on the cell side (IRN-78from Rohm and Haas or TRACOR Inc.).

GLC Columns and Column Packings

GLC packings are denoted as follows. The “3% OV-17” packing was 30 g of OV-17 per kilogram of thesupport, 100/120 mesh Chrom Q (Applied ScienceLaboratories, Inc., State College, Pa. 16801). The “4%

OV-17” packing was 40 g of OV-17 per kilogram of thesupport, Chromsorb G AW DMCS 80/100 mesh HP

(Analabs, New Haven, Conn. 06510). Poropak Q-S,100/120 mesh, was obtained from Waters Associates,Milford, Mass. 01757. All GLC columns were glass, 180

cm X 2 mm i.d.

Other Chemicals

All chemicals were AR grade. Drugs for use as stan-dards were obtained from pharmaceutical manufac-turers. Tolylbarbital (5-ethyl-5-p -tolylbarbituric acid)was obtained from Aldrich Chemical Co., Inc., Mil-waukee, Wis. 53233.

Methods

Extraction and GLC quantitation of ethchlorvynol

in plasma. Five milliliter aliquots of plasma containing20 mg ethchlorvynol per liter were extracted at pH 7

with differing volumes of n-hexane, in Teflon-lined,screw-capped extraction tubes. The extract was directly

analyzed by GLC (2-id injection) or, alternatively, wasfirst dehydrated with anhydrous sodium sulfate and

then analyzed by GLC without concentration of thehexane. GLC was done on a column containing 3%OV-17, at an injector and oven temperature of 125 #{176}Cisothermal (ethchlorvynol retention time, 2.5 mm). TheE1CD was operated in the reductive mode, with use of

Ni catalyst and a furnace temperature of 820 #{176}C.The

conductivity electrometer (range, attenuation) was 100

X 2 (100 mho X 2).Extraction and GLC quantitation of methaqualone

in plasma. One milliliter of a saturated solution of so-dium monobasic phosphate added to 1 ml of plasmacontaining 18 mg of methaqualone per liter was ex-tracted with 0.5 ml of n-hexane, and the extract wasprocessed as discussed above. GLC was on a 4% OV-17column with an injection temperature of 300 #{176}Candcolumn temperature of 275 #{176}Cisothermal. Methaqua-lone retention time was 8.0 mm. The E1CD was operatedin the reductive mode (H2 + Ni catalyst) at 830 #{176}C.

Plasma drug screening. Two milliliters of plasmacontaining 15 mg each of methyprylon, amobarbital,meprobamate, glutethimide, phenobarbital, andmethaqualone per liter was extracted with 25 ml ofchloroform at pH 7. The chloroform phase was isolated,dehydrated with anhydrous sodium sulfate, decanted,and evaporated. The residue was redissolved in 5O-tlof methanol and analyzed by GLC on 3% OV-17 (in-jector temperature 250 #{176}C,oven temperature pro-grammed from 125 to 300 #{176}Cat 8 0C/min). The E1CDwas operated in the reductive mode without catalyst,at a furnace temperature of 820 #{176}C.Comparative

chromatograms were obtained with use of 3% OV-17column interfaced to an FID system.

Effect of EICD furnace variables. The effects offurnace temperature and reaction gas on E1CD responsewere studied under defined conditions. The GLCstandard consisted of a solution of 1 mg!ml each ofmethyprylon, amobarbital, pentobarbital, secobarbital,meprobamate, glutethimide, phenobarbital, tolylbar-

bital, methaqualone, and diphenyihydantoin in meth-anol. A volume of 1 l was routinely used. GLC was done

742 (3JMCAI (HFMISTRV Vr,I IW 1Q7

on a 4% OV-17 column with an injector temperature of250 #{176}Cand with column temperature programmed from125 to 300 #{176}Cat 8 #{176}C!min.A quartz reaction tube (no

catalyst) was used to study the effect of furnace tem-perature (650 to 850 #{176}C)and reaction-gas conditions(per minute, 10 and 20 ml H2, 20 ml 02, pyrolytic) onE1CD response.

Extraction and GLC quantitation of meprobamate.

One milliliter of chloroform, containing 200 tg of p-dimethylaminobenzaldehyde, was added to 5-mi plasma

aliquots containing meprobamate at 40, 30, 20, 10, and0 mg/liter. Ten milliliters of chloroform was added and

the mixtures were extracted in Teflon-lined, screw-capped culture tubes. After the phases were separatedby centrifugation, the aqueous phase was aspirated, andthe chloroform phase was dehydrated with anhydrous

sodium sulfate, decanted, and evaporated. The residuewas redissolved in lOO-tl of methanol, and 2-id wasanalyzed by GLC on 3% OV-17, with an injector tem-perature of 225 #{176}Cand column temperature pro-grammed from 175 to 250#{176}Cat 8 #{176}C/min.The E1CD wasoperated in the reductive mode with Ni catalyst, a fur-nace temperature of 820 #{176}C,and electrometer settingof 300 tQ Xi.

GLC of volatile chlorinated hydrocarbons. Two-

microliter aiiquots of an aqueous solution of dichloro-methane, chloroform, and 1,2-dichioroethane (100 mg

of each per liter) were analyzed on a Poropak Q-S col-umn with injector and column temperatures of 190 and175 #{176}C,respectively, and E1CD electrometer set at 30tQ X 2. Comparative chromatograms were obtainedwith use of a Poropak Q-S column interfaced to an FIDsystem.

GLC of phenalkylamines. An injection of 100 ng eachof amphetamine and metamphetamine in 2-ii of n-hexane was analyzed by GLC on a 4% OV-17 column.The temperature of both the GLC injector and thecolumn was 125 #{176}C.The E1CD was operated in the re-ductive mode with use of a nickle catalyst, a furnacetemperature 825 #{176}C,and a conductivity electrometersetting of 100 pl X 1.

Extraction and GLC analysis of barbiturates inplasma. Plasma, 5 ml, containing about 15 mg each ofamobarbital, secobarbital, pentobarbital, and pheno-barbital per liter was extracted with 1 ml of isopropyl

ether in a Teflon-lined, screw-capped culture tube, andthe tube centrifuged to separate the phases; 2 tl of theisopropyl ether phase was analyzed by GLC on 4%OV-17 (injector temperature 250 #{176}C,oven temperatureprogrammed from 175 to 300 #{176}Cat 8 #{176}C/min).TheE1CD was operated in the reductive mode without cat-

alyst, furnace temperature 820 #{176}C,and an electrometersetting of 3 X 1.

Analysis of 1,4- benzodiazepines and 1,4- benzodi-azepin-2-ones. The 1,4-benzodiazepines (medazepamand N-desmethyl medazepam) and 1,4-benzodi-azepin-2-ones (diazepam, N-desmethyl diazepam, ox-

azepam, nitrazepam, flurazepam, N-i -hydroxyethylflurazepam, and N-1-desalkyl flurazepam) were ana-lyzed by GLC on a column containing 3% OV-17 with

an injector and column temperature of 235 #{176}Cisother-mal, The E1CD was operated in the reductive mode (H2+ Ni catalyst) at 850 #{176}Cfor qualitative analyses. A4-mm i.d. quartz reaction tube and Sr(OH)2 scrubber

were used in all analyses. The GLC/E1CD quantitativeanalysis of medazepam and diazepam and their major

metabolites in plasma was done by a modified proce-dure of de Silva and Puglisi (11). The procedure in-volves the extraction of 4-mi plasma standards (see

Results and Discussion) buffered to pH 9.0 into diethylether; the back extraction of drugs into 2 mol/liter HC1;

and final alkalinization of the acidic back extract fol-lowed by partitioning of drug into diethyl ether. Themodification consisted of the re-extraction of the al-

kalinized HC1 back-extract phase into chloroform ratherthan diethyl ether to avoid the requirement for the 30mm drying under reduced pressure to eliminate residual

water.Tricyclic antidepressants. Imipramine, desipramine,

amitriptyline, nortriptyline, protriptyline, and doxepenwere analyzed by GLC on a column containing 3% OV-17, with isothermal injector and column temperatureof 245 and 225 #{176}Crespectively. The E1CD was operatedin the reductive mode with Ni catalyst, 4-mm quartzreaction tube, Sr(OH)2 scrubber, and IRN-78/IRN-i50

stacked ion-exchange tube. The injection solvent wasroutinely vented prior to E1CD furnace, The extraction

of the tricyclic drugs from 4-mi plasma standards wasaccording to a modified procedure of Ervik and Ehrsson(12). Plasma standards of individual tricyclic drugs at

a concentration of 100-500 tg!liter were prepared withthe respective HC1 salts; 500 ng of promazine HC1 in-ternal standard was added to 5 ml of each plasma

standard; and these were made alkaline with 0.5 ml of1 mol/liter NaOH and extracted into 5 ml of benzene.The extraction tube was centrifuged, the benzene re-moved for evaporation, and the final residue redissolvedin 25 tl of benzene/methanol (19/1 by vol) for analysis

of 2 il by GLC/E1CD. Drug and internal standard werechromatographically resolved on OV-17, and the drugwas quantitated with the internal standard technique(see Results and Discussion).

Phenothiazines. Phenothiazine and the hydrochlo-ride salts of promazine, chlorpromazine, and triflu-operazine were analyzed by GLC/E1CD on a column

containing 3% OV-17 (injector and column temperature245 and 230 #{176}C,respectively). Nitrogen-selective de-

tection was achieved with H2 reaction-gas + Ni catalyst,and a Sr(OH)2 scrubber to remove acids such as HC1and HF. Furnace temperature was 850 #{176}C.

Results and DiscussionEthchlorvynol. Ethchlorvynol was quantitated by

direct extraction of plasma and GLC, with use of theHall E1CD in the reductive mode with nickle catalyst,and demonstrated selectivity and sensitivity at thenanogram level when hydrocarbon solvents are used(Figure 2). Additionally, GLC/EICD analysis withoutfurther cleanup or concentration of the organic extractsaves time and avoids analytical error arising from

ASSAYS

C

#{163}ILLL4Fig. 2. a.C-EICD chromatograms of ethchlorvynol standards andplasma extractionsCfromatograms A, B, C, and Dare for 10, 20, 100, and 200 ng of ethchlorvynolIn 2-SI of hexane. Chromatograms E, F, and Gate for direct GLC analysesofhexane extracts (10, 5, 1 ml of extract) of 100 ng of ethchlorvynol in 5 ml ofplasma

C

B

J.JFig. 3. GLC/EICD chromatogram of 100 ng of amphetamine (B)and methamphetamine (C) in 2 I of hexane (A)Furnace chemistry: reductive mode, with catalyst

CLINICALCHEMISTRY, Vol.22,No.6,1976 743

ST ItCH LON V SNOt.

CH3-c,,oHC=Cl “CCHH”

LL

possible volatilization of ethchlorvynol during solvent

evaporation. The analytical sensitivity necessary formicro-scale analysis can be achieved by a combinationof electrometer attenuation (200-fold increase avail-

able), optimization of furnace-chemistry variables suchas H2/He carrier gas ratio and decrease in conductivity

cell liquid-flow rate, to which detector response is in-versely proportional. Factors limiting cell flow-rate tonot less than 0.1 ml!min include peak broading (solute

diffusion) and the operation of the gas-liquid contactorand separator.

Methaqualone. Nitrogen-selective E1CD response

to methaqualone was linear over the range of 10-1000ng studied. The sensitivity and element-selectivity ofthe detector are reflected in lower detection limits,greatly enhanced drug-to-solvent peak response, andabsence of plasma interference peaks produced byplasma co-extractables. The extraction with bufferedhexane permits the use of smaller solvent volumes (lessemulsion) and direct GLC analysis because the relative

detector response to hydrocarbons in the reductivemode is very low.

A slight negative pen deflection seen immediatelypreceding the leading edge of the methaqualone peaksuggests a solvent-mediated neutralization reaction(NH3 + H3O - NH4 + H2O). Such a neutralizationphenomenon (conductivity solvent too acidic) will be

the factor limiting detectability unless a basic ex-change-resin bed mixture is used to maintain the pH ofthe conductivity solvent at greater than 7.

Meproba mate. Quantitation of meprobamate inplasma by GLC/E1CD by use of p-dimethylaminoben-zaldehyde as the internal standard, required the use ofH2 reaction gas and nickle catalyst. Chromatogramsdemonstrated a linear E1CD response, the eliminationof significant interference from co-extractables, andmore definitive qualitative identification (i.e., based onboth retention time and element-selectivity) as com-

pared to FID analysis. Additionally, change in relativedetector response (meprobamate vs. internal standard)with different furnace chemistries can be used to con-

firm qualitative identification. For example, withoutnickle catalyst, the primary amino groups of meproba-mate are still detected as NH3 via a reductive decom-position mechanism at 820 #{176}Cfurnace temperature,

while the tertiary amine of the internal standard pro-duces no E1CD response unless nickle catalyst is used.Thus, GLC retention time, E1CD element-selectivity,and relative E1CD response together with the use ofappropriate furnace chemistries provide a set of com-plementary analytical data that allow more nearly

certain qualitative identification and quantitation.However, GLC/FID analysis produces questionabledata when nonselective extraction and cleanup proce-dures are used.

The choice of E1CD furnace chemistry or internalstandard for drug quantitations requires considerable

care until more is known about the principles of detectorresponse. In addition to the choice of an internal stan-

dard based on mutual co-extraction with the drug ofinterest and their adequate resolution and detection by

chromatographic instrumentation, principles of E1CDresponse and detector variability should be considered.

Variations in E1CD that may cause nonlinearity in de-tector response ratio with differing concentration ratios

of drug and internal standard include: (a) specific re-quirement for furnace chemistry mode, which may af-fect the working linear range of drug or internal stan-dard detection; (b) gaseous or solution-phase reactivityof furnace products (e.g., NH3 + HC1 or SO); (c) dif-ferential effects of post-furnace abstractors; and (d)

fluctuations in pH of conductivity solvent that differ-

ently affect dissociation constants of furnace reactionproducts from drug and internal standard. These de-tector variables must be considered rather distinctly

0

744 CLINICAL CHEMISTRY, Vol. 22. No. 6, 1976

from those traditionally encountered in GLC/FIDanalysis-i.e., different relative extraction efficiencies

at different drug/internal standard concentration anddifferential thermal or chromatographic decomposition.

Criteria for choosing an internal standard to be used inGLC/EICD analysis include: (a) compounds containingonly the one reactive element (N, halogen, 5) presentin the drug to be quantitated; (b) compounds with

functional group chemistry as similar to that of the drugof interest as possible; and (c) compounds with an op-timum furnace chemistry similar to that of the drug

being measured. The quantitation of drugs with two ormore reactive elements (e.g., phenothiazines) or theneed to use reaction gas abstractors may present addi-tional analytical problems, and these problems are beingactively considered in this laboratory.

Phenalkylamines. E1CD sensitivity and selectivity(relative to hydrocarbons) for nitrogen-containingphenalkylamine congeners was demonstrated by the

analysis of 100 ng each of amphetamine and metham-phetamine in a 2-id hexane injection (Figure 3). Hall(13) has successfully detected 5 ng of amphetamine in

combination with 10 ng of methamphetamine on acolumn containing alkaline Carbowax, with a good

signal/noise ratio. He used the E1CD reductive modewith nickle catalyst, but substituted electrolytic-grade

hydrogen for helium as the carrier gas.With GLC!E1CD, sensitivity is a function of chro-

matographic phenomena (decomposition, sorptionprocesses, peak shape, etc.), detector variables, cellchemistry, and residual detector noise. The choice ofE1CD furnace-chemistry variables such as reaction tube,reaction catalyst, reaction gas, furnace temperature, andreaction product abstractors is critical to maximizingdetector sensitivity. These variables affect conversion(reaction products of the drug) and throughput (theproportion of the reaction products that is delivered tothe conductivity cell for a particular furnace chemistrymode and compound). Conversion rate is not only de-termined by the decomposition mechanisms operativewith a particular furnace chemistry, but also by theresidence time of the GLC effluent in the reaction tube.Sorption-desorption processes involving reaction tubeand nickle catalyst surface phenomena may also playa minor role in affecting conversion-and-throughput

and peak shape, as do the physical configuration andchemistry of acidic or basic abstractors in the post-furnace reaction tube. Contamination from solvent ordrug carbonization processes or GLC “bleed” in thereaction tube or post-furnace transfer line will increasedetector background and may trap furnace reactionproducts. A consideration of cell chemistry must includethe processes of effective gas-liquid mixing-solubili-zation-and ionization, which may limit trace analysis.Mixing and solubilization may be improved by using low

flow rates for the conductivity solvent (0.1-0.5 ml/min)to both increase contact time and increase effectiveconductance. Optimum solvent pH is important toprevent neutralization reactions, while solvent impurity

and contamination from the ion-exchange bed will af-

Fig. 4. GLC/EICD chromatogram of isopropyl ether extract ofamobarbital (A), pentobarbital (B), secobarbital (C), and phe-nobarbital (D) in plasma, 15 mg of each per liter

feet background conductivity. A quantitative discussionof some detector variables and residual detector noise

has been presented by Hall (8, 9).

Barbiturates. Figure 4 shows the GLC/E1CD chro-matogram for a 2-id injection of the isopropyl ether

extract from a sample of plasma containing 15 mg of theindicated barbiturates per liter. If one assumes 100%

extraction, each peak represents less than 75 ng of drug.Differences among these compounds in the percentage

content of nitrogen or their relative extractability do notexplain the dramatic differences in detector response,which more probably are attributable to the furnacechemistry of the individual drugs.

To study furnace chemistry and detector response,

we varied E1CD reaction gas conditions while holding

constant the conductivity solvent flow-rate, solvent pH,and furnace temperature. Use of the reductive modegreatly improved peak resolution. With decreased H2reaction gas glow rate, detector response became in-creasingly similar to that obtained under pyrolyticconditions. Under reductive conditions, methaqualonewas not detected without the use of a nickle catalyst.The inability to detect diphenylhydantoin under the

conditions noted was due to short reaction-tube resi-dence time; this may be corrected by using a larger (4mm i.d.) reaction tube.

The effect of E1CD furnace temperature on detectorresponse and peak resolution was studied under re-ductive conditions (H2, no catalyst). Peak shape and

peak height were optimum with an 800-850 #{176}Cfurnacetemperature. At present, the recommended furnacechemistry for barbiturate screening is 20 ml of H2 re-action gas per minute, no catalyst (barbiturates poisonnickle catalyst), and furnace temperature of 820 #{176}C.Figure 5 shows a chromatogram obtained from ainjection of a 1 mg/ml methanolic drug mixture withE1CD, at the recommended furnace and detector set-

tings.

Plasma drug screening. Figure 6 demonstrates thecomparative advantage of E1CD over FID in the GLCscreening of serum or plasma for weakly acidic andneutral drugs. Chromatogram A illustrates reducedE1CD response to co-extractable interferences and the

IA

IIc

E

B

LiL

bC

B

Fig. 6. Comparative GIC/FID (upper) and GLC/EICD (lower)chromatograms of plasma extracts containing methyprylon (A),amobarbital (B), meprobamate and glutethimide (C), pheno-barbital (0), methaqualone (E), phthalate ester (F), and uniden-tified plasma co-extractable (?)Analyses Include: chromatogram A, routine plasma extraction; chromatogramB, plasma in contact with “serum skimmer”; chromatogram C. extraction of“serun#{149}Iskimmer”

CLINICAL CHEMISTRY, Vol. 22, No. 6. 1976 745

Fig. 5. GLC/EICD chromatogram (reductive mode, no catalyst)of acidic-neutral drug mixtureMethanol solvent front (A), ethchlorvynol (B), elhosuximlde (C), metharbital (0),barbital (E), methyprylon (F), butabarbital (G), amobarb’rtal (I. pentobarbital (0.secobarbltal (,, glutethimide (5), mephobarbital (L), phenobarbital (M), andtolylbarbital (M

capability of obtaining a more nearly accurate quanti-tative analysis. Additionally, there are fewer misinter-pretations of “serum peaks” as unidentified drug con-

stituents. The FID response in chromatogram B dem-onstrates an additional interference from a phthalateester (identified by mass spectrometry), caused bycontact of the plasma with a commercial “serum skim-mer” for 20 mm before the extraction. This contaminantwould make quantitation of methyprylon by FID moredifficult. Chromatogram C, another dual FID/E1CDtracing obtained from a chloroform extract of the“serum skimmer”, shows no E1CD response to the car-

bon-, hydrogen-, oxygen-containing phthalate ester.

ER.d

Li3 i

F IDjJ

EOxld

! UjJ j U JFUIP4AC( ITEMP 1000#{176}C. 950#{176} o#{176} I #{149}54#{176}

(IC) ISC- Nttcatalyst)

i#{252}LEC-R.d,Ni

D C I A

LL iJu IU IFig. 7. Comparative GLC chromatograms of dichloromethane(1), chloroform (2), and dichloroethane (3) by electrolytic con-ductivity (EC) and FIDEICDfurnace modes Included:reductive wIthout catalyst (EC-Red); oxidativewithout catalyst (EC-Oxhi); pyrolytic with catalyst (EC-Ni); and reductive withcatalyst (EC-Red, NO. E1CDfurnace temperatures were 850 (A), 900 (B), 950(C),and 1000 #{176}C(D)

Volatile chlorinated hydrocarbons. Volatile chlori-nated hydrocarbon chromatograms obtained frommatched Poropak Q-S columns are summarized in

Figure 7. Variations in E1CD furnace chemistry are re-flected in changes in relative detector response betweendichloromethane:chloroform:dichloroethane and total“chlorine response”:water peak as a function of reac-tion-gas composition, catalyst, and furnace tempera-ture. In addition to the improved sensitivity and selec-tivity of E1CD for halogenated volatiles compared toFID, GLC/E1CD improves the certainty of correctqualitative identification by allowing the use of a type

of “pattern recognition.” This “pattern recognition”refers to the comparative change in detector responsefor the unidentified peak as compared to that for acholorinated internal standard with change in furnacechemistry. Although Dolan and Hall (8) have com-mented on dechlorination mechanisms for polychiori-nated systems, the elucidation of the competitive de-composition mechanisms involved in these chlorinatedaliphatic systems is incomplete.

Figure 8 demonstrates a potential application ofE1CD vs. FID with regard to the relative selectivity inthe analysis for chlorinated volatiles. ChromatogramsA are matched FID and E1CD detector response to a

2-Ml injection of a 100 mg/liter solution of chlorinated

Sn

C

A

1jD

2

jJFig. 11. GLC/EICD chromatograms of medazepam (A), N-des-methyl medazepam (B), diazepam (C), and N-desmethyl di-azepam (0), demonstrating the effect of neutralization reactionsand Sr(OH)2 scrubberFurnace chemistry: reductive mode, with catalyst and Sr(OH)2 scrubber. Chro-matogram 1, 100 ng A-D, electrometer 100 X 2 attenuation, and 10mgof scrubber. Chromatogram 2, 20 ng A-D, electrometer 30 X 1 attenu-ation, and 10mg of scrubber. Chromatogram 3,2 ng A-D, electrometer 3 Lu1X 1 attenuation, and 10 mg of scrubber. Chromatogram 4. 2 ng A-D, elec-trometer 3 I& X 1, and 20mg of scrubber. Arrow indicates venting of Injectionsolvent. () indicates neutralization reaction causing negative peak

A

.911

AB

F

ItwERATURE

Fig. 10. Graphical representation of the effect of EICD furnacetemperature on detector response (peak height) for the injectionof 100 ng of medazepam (A), N-desmethyl medazepam (B),diazepam (C), N-desmethyl diazepam (D), oxazepam (E), flu-razepam (F), N-l-desalkyl flurazepam (0), N-1-hydroxyethylflurazepam (H), and nitrazepam (I)Furnace chemistry: reductive mode, with catalyst and Sr(OH)2 scrubber

748 CLINICAL CHEMISTRY, Vol. 22, No. 6, 1976

FID H

EC-Red.

32 1

Fig. 8. GLC/EICD and GLC/FID chromatograms of dichloro-methane (1), chloroform (2), and dichloroethane (3)Chromatogram A, 2 tI of 1, 2, and 3(100 mg/liter). Chromatogram B, 2 M’ ofethanol (4) and n.propanol (5) (800 mg/liter). Chromatogram C, 2 l of 1, 2, and3 (50 mg/liter) in solution with 400 mg of 4 and 5 per liter

G C

F

B

Fig. 9. GLC/EICD chromatograms of 50 ng of medazepam (A),N-desmethyl medazepam (B), diazepam (C), N-desmethyl di-azepam (D), oxazepam (E), flurazepam (F), N-1-desalkyl flur-azepam (G), N-1-hydroxyethyl flurazepam (H), and nitrazepam(I)Furnace chemistry: reductive mode, with catalyst and Sr(OH)2 scrubber. Arrowsindicate the venting of injection solvent

Vj.

volatiles in water. Chromatograms B demonstrate theselectivity of E1CD (reductive mode, no catalyst) againstaliphatic alcohols (ethanol and n-propanol, 800 mg/liter,2-,l injection). Matched FID and E1CD chromatograms

C of a 2-id injection of 400 mg/liter each of ethanol andn-propanol in solution with 50 mg/liter chlorinatedhydrocarbons illustrates that E1CD affords a more

nearly certain qualitative identification and quantita-

tion.1,4-Benzodiazepines and 1,4-berizodiazepin-2-ones.

The sensitive nitrogen-selective analysis of thesehalogen- and nitrogen-containing congeners required

the use of a 4-mm i,d. reaction tube to allow longer re-action-tube residence time and the use of a Sr(OH)2scrubber to remove HC1 and HF reaction products thatwould otherwise result in peak tailing and neutralizationreactions (e.g., gas-phase: NH3 + FIX - NH4X; solu-

tion-phase: NH4 + OH- + HX -* NH4 + X + H20).Under the prescribed E1CD furnace and GLC condi-tions (see Methods), a cell flow-rate of 0.5 ml/min, and

electrometer setting of 100 tQ1 X 1, the on-columninjection of 50 ng of drug is easily detected (Figure 9).With these GLC and cell flow-rate conditions, theminimum detectable quantity with a signal/noise ratio

of 2/1 was 500 pg of medazepam (A), 500 pg of N-des-methyl medazepam (B), 1 ng of diazepam (C), 20 ng ofN-desmethyl diazepam (D), 2 ng of oxazepam (E), 2 ng

of flurazepam (F), 2 ng of N-1-desalkyl flurazepam (G),15 ng of N-1-hydroxyethyl flurazepam (H), and 20 ng

of nitrazepam (I).A lower minimum detectable quantity may be

achieved by decreasing cell flow-rate, optimizing the H2reaction-gas flow-rate, and increasing furnace tem-

perature (see Figure 10 for temperature effect). A lower

AB

C

D

A5

“4

0

0

0

4

4ELIa

B

A

CLINICALCHEMISTRY, Vol.22,No.6, 1976 747

Fig. 12. GLC/EICD chromatograms of extracts of medazepam,diazepam, and metabolites in plasmaFurnace chemistry: reductive mode, with catalyst and Sr(OH)2scrubber. Chro-matogram 1, analysis of 4 mi of plasma containing 200g medazepam (A), 300tg of N-desmethyl medazepam (B), 40 g of diazepam (C), and 80 tg of N-desmethyl diazepam (0) per liter; electrometer 30 z1-’ X 1 attenuation.CPwomatogram 4, analysis of 4 ml of plasma containing 40 g of internal standardA, 40g ofC, and 80g of0 per liter; electrometer 3 dl X 1 attenuation.Arrow indicates venting of Injection solvent. Lower chromatogramsrepresentthe analysis of blank plasma

minimum detectable quantity for congeners such as B,D, E, G, H, and I might also be realized by derivatizationto improve GLC volatility and peak shape, and to re-

duce adsorption losses (14). The quantity of Sr(OH)2scrubber used also affects the minimum detectablequantity (Figure 11). The “operative” phenomena in

determining the detector’s minimum detectablequantity include the relative production of HC1 or HFvs. NH3 under a specific set of reaction conditions, thetrapping efficiency of Sr(OH)2 for HX, and thethroughput of NH3 and HX to the conductivity cell. Forexample, chromatograms 3 and 4 in Figure 11 demon-strate the importance of the scrubber in trapping HC1and preventing neutralization reactions.

The quantitative analysis of medazepam and di-azepam, and their major biotransformation productspresent in plasma, was done by the modified extractionmethod of de Silva and Puglisi (see Methods) and

GLC/E1CD (Figure 12). Chromatogram 1 is theGLC/E1CD analysis of a 4-ml plasma extract containing

200 g of A, 300 tg of B, 40 g of C, and 80 g of D perliter. Based upon reported recovery efficiencies (11), the

chromatogram represents the on-column injection ofapproximately 60 ng of A, 90 ng of B, 12 ng of D, and 24ng of D. The quantitative analysis, as judged byGLC/E1CD, was linear over at least a 10-fold increasein drug concentrations. Chromatogram 4 illustrates theanalysis of 4 ml of plasma containing 40 g of C and 80ig of D per liter. The analysis of plasma resulted in nosignificant interference peaks. The quantitation ofmedazepam, diazepam, and metabolites in plasma ac-cording to the more straightforward protein precipita-tion/extraction/direct GLC analysis of de Silva et al.(14) using E1CD also resulted in chromatograms free ofinterference peaks and allowed the quantitation of

therapeutic drug concentrations.

100 200 300 400 500

DRUG CONC., CS/mi

Fig. 13. Standard curves of the GLC/EICD response to theanalysis of extracts of plasma containing various concentrationsof imipramine (A), desipramine (B), amitriptyline (C), nortriptyline(0), protriptyline (F),and doxepen (F), with 500 ng of promazineHCI as internal standard per 4 ml of plasma analyzedFurnace chemistry: reductive mode, with nickle catalyst and Sr(OH)2 scrubber

Fig. 14. GLC/EICD chromatograms of 20 ng of phenothiazine(A), 32 ng of promazine HCI (B), 35 ng of chlorpromazine HCI(D), and 48 ng of trifluoperazine 2HCI (0)Furnace chemistry: reductive mode, wIth catalyst and Sr(OH)2 scrubber; elec-trometer 10 1zt)’ X 2 attenuation. These quantities of A-Drepresent 1.4. 2.8.2.8, and 4.2 ng of nitrogen, respectively

A comparison between nitrogen- and chlorine-se-

lective E1CD and electron capture analysis for thesedrugs and drug metabolites is presently underway in

this laboratory.Tricyclic antidepressants. The quantitative analysis

of tricyclic drugs in plasma was linear over the 100-500tg/liter concentration range (Figure 13). Differences inthe individual calibration curves representing E1CDresponse to drug vs. internal standard are due to theweight-percent of nitrogen in the individual compounds,the furnace chemistry mechanisms resulting in NH3production, and relative efficiencies of extraction and

GLC. Although of about the same molecular weight,imipramine (A) and desipramine (B) contain a nitrogen

in the ring structure not present in C-F. A ranking ofE1CD response (A > B > C F> D > E) to equimolarinjections of these tricyclic compounds at optimizedfurnace conditions demonstrated that E1CD response

to N,N- dimethyl analogs was greater that the response

748 CLINICALCHEMISTRY,Vol.22,No. 6,1976

to their respective N-methyl congeners (e.g. A > B andC > D). E1CD response to protriptyline was the lowestbecause of both its weight-percent nitrogen and thefurnace chemistry associated with its secondaryalkylamino substituent. Further differences in thequantitative analytical response (Figure 13) to equiv-

alent drug concentrations of A and C compared to theirN-methyl analogs B and D is most probably due to the

greater adsorptive loss of B and D during extraction andGLC.

Drug and drug metabolite combinations such as A-B

and C-.D are not resolved by GLC on a column con-taining OV-17. However, derivatization with aceticanhydride will allow separation by GLC (retentionorder: N,N- dimethyl analog, promazine, N-methylacetamide derivative) and accurate quantitation at drug

concentrations of greater than 75 tg/liter of plasma.Nonlinearity at low drug concentrations is due to lossin extraction, GLC, and E1CD reaction-product

throughput to the conductivity cell. These phenomenaare being investigated in an attempt to develop a reli-able quantitative analysis in the 5-40 gig/liter concen-tration range encountered in the therapeutic monitoring

of tricyclic antidepressant drug concentrations.Phenothiazines. Phenothiazines analyzed by

GLC/E1CD were detected in the nitrogen-selectivemode (Figure 14). Detector response was quantitativelyrelated to the nitrogen content and did not show thevariations caused by side chain chemistry that werenoted for the tricyclics. The minimum detectablequantity of phenothiazine was less than 100 pg.

A quantitative comparison of the element-selectivityof the E1CD in the nitrogen-, halogen-, and sulfur-se-lective modes vs. flame photometric and electron cap-

ture detectors is being undertaken by this laboratory.

I conclude that the Hall E1CD is a more sensitive andelement-selective detector than FID, and permits GLCanalysis with less sample clean-up, direct injection ofsample extracts without concentration of the organicsolvent, and a more certain qualitative identification.Various E1CD furnace chemistry modes permit thedetection of organic nitrogen, halogen, sulfur, or carbon,

as contrasted to other less flexible element-selectivedetectors (e.g., flame photometric-sulfur and phos-phorus, thermionic or alkali flame-nitrogen or phos-phorous, and electron capture-halogens). The current

cost of the E1CD ($2995) is not prohibitive, and isavailable as part of a complete GLC/E1CD instrument(TRACOR Inc.) or it may be interfaced with most com-mercial gas chromatographs.

I thank Ken Mahier, of TRACOR Inc., for support of this study, and

Timothy Flynn for technical assistance.

References1. McNair, H. M., and Bonelli, E. J., Basic Gas Chromatography, 5thed., Varian Aerograph, Walnut Creek, Calif., 1969, pp 118-119.

2. Hartman, C. H., Gas chromatograph detectors. Anal. Chem. 43,124A (1972).

3. Toseland, P. A., Albani, M., and Gauchel, F. D., Organic nitro-gen-selective detector used in gas-chromatographic determinationof some anticonvulsant and barbiturate drugs in plasma and tissues.Clin. Chem. 21,98 (1975).

4. Piringer, 0., and Pascalau, M. J., Em neuer Detektor fur die Gas-chromatographie. J. Chromatogr. 8,410 (1962).

5. Coulson, D. M., Electrolytic conductivity detector for gas chro-matography. J. Gas Chromatogr. 3, 134 (1965).

6. Jones, P., and Nickless, G., Versatile electrolytic conductivitydetector for gas chromatography. J. Chromatogr. 73, 19 (1972).

7. Selucky, M. L., Specific gas chromatography detectors. Part II:Electrolytic conductivity detector. Chromatographia 5, 359 (1972).

8. Dolan, J. W., and Hall, R. C., Enhancement of the sensitivity andselectivity of the Coulson electrolytic detector to chlorinated hydro-

carbon pesticides. Anal. Chem. 45, 2198 (1973).

9. Hall, R. C., A highly sensitive and selective microelectrolyticconductivity detector for gas chromatography. J. Chromatogr. Sci.12, 152 (1974).

10. Product description: Model 310 Hall electrolytic conductivitydetector (TRACOR Inc., Austin, Tex. 78721).

11. de Silva, J. A. F., and Puglisi, C. V., Determination of medazepam(Nobrium), diazepam (Valium) and their major biotransformationproducts in blood and urine by electron capture gas-liquid chroma-tography. Anal. Chem. 42, 1725 (1970).

12. Ervik, M., Walle, T., and Ehrsson, H., Quantitative gas chroma-tographic determination of nanogram levels of desipramine in serum.Acta Pharm. Suec. 7, 625 (1970).

13. Hall, R. C., Retention Times (TRACOR Bulletin), TRACOR Inc.,Austin, Tex. 78721 (1974).

14. de Silva, J. A. F., Bekersky, I., Puglisi, C. V., et al., Determinationof 1,4-benzodiazepines and -diazepin-2-ones in blood by electron-capture gas-liquid chromatography. Anal. Chem. 48, 10 (1976).