Journal of Automatic Chemistry, Volume 6, Number (January-March 1984), pages 14-20

Program in BASIC to combine datafrom two different selective detectorsand its application for screening ofpesticides in residue analysisH.-J. Stan and H. GoebelInstitut fFtr Lebensmittelchemie der Technischen Universittt Berlin, Miiller-Breslau-Strafle, 1000 Berlin 12, FR Germany

Introduction

For the analysis of pesticide residues in food, gas chroma-tography with selective detectors is established internationallyas the most suitable method. The application ofelectron capture(ECD) and nitrogen-phosphorus (NPD) detectors enables theselective detection of contaminants at trace level in the presenceof a multitude of compounds extracted from the matrix, whichdo not respond to these detectors.

The number of compounds used in agriculture for plantprotection and the variety of pollutants in the environment hasincreased to such a level that it is impossible to separate them allin a single chromatogram, despite using high-performancecapillary columns. These capillary columns allow the retentiontimes ofcompounds to be determined with a very high accuracyand good reproducibility. The high resolution facilitates thedifferentiation of substances belonging to the same structuralclass as organophosphate pesticides (PP) or chlorinatedpesticides (CP).

When splitting the effluent of the capillary column to bothselective detectors, additional information about the identity ofthe individual compounds can be obtained from the chromato-grams by calculating the response ratios.

A concept has recently been developed for automatedpesticide residue analysis--realizing it by means of a gaschromatograph with options for BASIC programming, dual-channel operation and automatic liquid sampling [1 and 2].

The BASIC program, which controls the automatedanalysis, calculates the residue concentrations and evaluates thedegree of certainty of the pesticides found is presented here.

Materials and methods

The pesticide residue analysis was performed with a gaschromatograph (HP 5880 A, Hewlett Packard, Palo Alto,California, USA) using capillary columns (BP 1, SGE-ScientificGlass Engineering, Australia) and effluent splitting to the twoselective detectors (NPD and ECD). The signals from detectorsare processed in a dual-channel integrator connected to twoterminals, allowing both chromatograms to be recorded inparallel. The manufacturer’s operating system and chroma-tography progfams are used for data collection, immediaterecording ofthe chromatograms, recognition ofcalibrated peaksand quantitation. The internal standard method is generallyused. The instrument is also equipped with an autosampler, HP7671A, and the microprocessor’s memory is extended by acartridge tape unit.

The procedure of the complete pesticide analysis has alreadybeen described [1 and 2].

14

The program

The HP 5880A was launched as the first gas chromatographwhich could be programmed in BASIC; this allows it to executeindividual calculations, to format reports and to control theautosampler. The authors’ program was developed to fulfil thefollowing requirements:

(1) The ability to run a food sample with parallel signalprocessing in two channels and storage of the data.

(2) Calculation of peaks found by means of several cali-bration tables.

(3) Evaluation of the results by comparing the peaksidentified in the two channels.

(4) Summarizing final results in a clearly arranged report.

The HP 5880A was designed to store one calibration table ineach channel. However, the large number of pesticides includedin the authors’ analytical method requires at least three cali-bration mixtures with the three corresponding calibration tablesin each channel. Therefore the calibration tables have to besaved on an external memory: in this case a tape. The calibrationtables are included in the three ’Analysis files’. Each file containstwo calibration tables generated by dual-channel recording ofone calibration mixture. Additionally, the analysis files containthe parameter settings of the instrument to run the gaschromatographic analysis. A special problem arises from thefact that communication between the two channels is limited--calculations can easily be performed, but processing in onechannel cannot be controlled by the other one. Therefore twoseparate programs have been created, these are synchronized bymeans of waiting loops.

The programs are docu.,rnented in figures and 2; the REMstatements should make them almost self-explanatory.

For readers unfamiliar with the HP 5880 A, a list of some ofthe commands for activating special procedures and functionsintegrated in the program might be helpful:

START AUTO SEQ X, X: Starts the automatic sampler.The two numbers define the first and last bottle.RECALIB: The areas from the calibration mixtures foundin the most recent run and the amounts from the originalcalibration run of the same mixture are used to calculatenew response factors.RECALIB RUN TIME: The retention times in the cali-bration table are replaced by real times found in the mostrecent run.SAMPLE $: Returns the number of the sampler bottle inthe tray.ID $: Returns the sample names from the sample table.

H.-J. Stan and H. Goebel BASIC program to combine data from two selective detectors

DETECTOR A O: NPD is switched off.AMT (I)" Returns the concentration of a specified peakcalibrated.HEAD $: Returns the title from the calibration table.

Application to a real food sampleThe automated pesticide residue analysis controlled by theBASIC program described has been designed as a screeningprocedure. As already mentioned, a complete residue analysis infood of unknown origin cannot be performed on a singlecapillary column: so the aim of automated screening is toprovide the analyst with information about contaminants that

may be present in the specified sample. All of the suspectedpesticides listed in the final report are analysed using anindependent method of confirmation [1 and 2]. Examples offinal reports from the two channels are shown in figures 3 and 4.All peaks identified by means of the data stored for eachcalibration mixture are printed, and those responding to bothdetectors are compared by applying their specific responseratios. The deviation from the value calculated from thecalibration data is reported as a percentage. A difference ofmorethan 30 is usually an indication that the peak found does notcorrespond to the substance calibrated. Also, all compoundsidentified with only one detector are indicated, as well as thosebelonging to ’critical pairs’ in chromatography and thoseresponding to both detectors.

LIST PRGMPROGRAM:

102030405O60708O901001101201301401501601701801902OO2102202302402502602702802903OO31032033034O35036037038O3904OO41042043044045O4604704804905O051052O53O54O55O56057058O590600

(ANNOTATION OFF)PRINT "LEADING PROGRAM (CHANNEL 1) FOR RESIDUE ANALYSIS"PRINT "PARALLEL IN NPD AND ECD"REMREM ********** NPD (SIGNAL A): CHANNELREM ********** ECD (SIGNAL D): CHANNEL 2REM ********** COLUMN: METHYLSILICONE- BP1REM ********** INTERNAL STANDARDS FOR NPD: NT (PT)REM ********** FOR ECD: ALDRIN (1,2,3- TCB)REM ********** CALIBRATION TABLES AND INSTRUMENT SETPOINTSREM ********** IN ANALYSIS FILES 1,2 AND 3PRINTPRINT "PUSH Y IF LOADING THE THREE STANDARD MIXTURES INTO THE TRAY"INPUT "AS FOLLOWS: PPI, PPII, CPI AND STARTING CHANNEL 2", A$IF A$ < > "Y" THEN 120PRINTPRINT "CREATION OF SAMPLE TABLE"PRINTPRINT "START WITH 4 AND CONCLUDE BY PUSHING EXIT"PRINTSAMPLE TBLINPUT "HOW MANY BOTTLES ARE IN THE TRAY?",NLET C N-3GET ANALYSIS DEVICE* 6OVEN TEMP ANNOTATION OFFLIST CLOCK TIMESTART AUTO SEQ 1,1RECALIBRECALIB RUN TIMEWAIT 0.2DELETE ANALYSIS DEVICE* 6SAVE ANALYSIS DEVICE* 6GOSUB 2030GET ANALYSIS 2 DEVICE* 6OVEN TEMP ANNOTATION OFFSTART AUTO SEQ 2,2RECALIBRECALIB RUN TIMEWAIT 0.2DELETE ANALYSIS 2 DEVICE* 6SAVE ANALYSIS 2 DEVICE* 6GOSUB 2030GET ANALYSIS 3 DEVICE* 6OVEN TEMP ANNOTATION OFFSTART AUTO SEQ 3,3RECALIBRECALIB RUN TIMEWAIT 0.2DELETE ANALYSIS 3 DEVICE* 6SAVE ANALYSIS 3 DEVICE* 6GOSUB 2030WAITGET ANALYSIS DEVICE* 6REMREM ********** INJECTION OF C SAMPLES AND STORING THE DATA ON TAPEREMFOR Nl=4 TO NOVEN TEMP ANNOTATION OFFATTN 2T2THRESHOLDSTART AUTO SEQ N1,N1

15

H.-J. Stan and H. Goebel BASIC program to combine data from two selective detectors

61062063064065066067068069070071072073074075076077078079080081082083084085086087088089090091092093094095O9609709809901000101010201030104010501060107010801090110011101120113011401150116011701180119012001210122012301240125012601270128012901300131013201330134013501355136013701380139014001410

PRINT SAMPLE$,ID$LIST SIGNALWAIT 0.5EXECUTE E, "SAVE REPORT "&AL$(N1)&" DEVICE* 6"NEXT N1PRINTDETECTOR A 0PRINTPRINT TAB(15); "REGISTER OF PESTICIDE RESIDUES OF"; C; "SAMPLES"PRINTPRINTIMAGE X,2A,4X,12A,4X,12A,4X,DD.DD,4X,DD.DD,4X,DD.DD,4X,DDDDDDIMAGE X,2A,4X,12A,4X,12A,4X,5A,4X,5A,4X,6A,4X,6APRINT USING 730; "NO", "SAMPLE", "NAME", "PPM", "RT", "EXP.RT", "AREA"PRINTPRINTFOR Nl=4 TO NPRINT "CALCULATED FROM STANDARD MIXTURE FOR P-COMPOUNDS"PRINTWAIT 0.5REMREM ********** LOADING THE REPORTS INTO MEMORY AND COMPARISON WITH.REM ********** THREE STANDARD MIXTURESREMEXECUTE E, "GET REPORT "&VAL$(N1)&" DEVICE* 6"GOSUB 1040WAITGET ANALYSIS 2 DEVICE# 6PRINTPRINT "CALCULATED FROM STANDARD MIXTURE II FOR P-COMPOUNDS"PRINTGOSUB 1040WAITGET ANALYSIS 3 DEVICE* 6PRINTPRINT "CALCULATED FROM STANDARD MIXTURE FOR N-COMPOUNDS"PRINTGOSUB 1040GOTO 1320REMREM ********** PRINTING REPORTS AND COMPARISON OF RESULTSREM ********** BETWEEN ECD AND NPDREMFOR TO *PEAKSIF AMT(I)> =0.01 THEN 1070GOTO 1080PRINT USING 720; SAMPLES, IDS, NAME.S(I),AMT(I),RT(I),EXPRT(I),AREA(I)NEXTPRINTFOR I- TO *PEAKSLET US NAMES(I)LET F AMT(I)FOR J= TO *PEAKS(2)IF AMT(I)<0.01 THEN 1290IF NAMES(J,2)= U$ THEN 1170GOTO 1280IF AMT(J,2)> =0.01 THEN 1190GOTO 1280LET E AMT(J,2)IF F > E THEN 1220IF E> F THEN 1240LET Y-(F-E).100/FGOTO 1250LET Y (E-F). 100/EIMAGE "THE DIFFERENCE BETWEEN THE DETECTORS AMOUNTS TO",DDD,""PRINT USING 1250;YPRINT "FOR",NAME$(J,2)NEXT JNEXTPRINTRETURNIMAGE 72(’’’)PRINT USING 1320PRINTGOSUB 1580WAITGET ANALYSIS 2 DEVICE* 6GOSUB 1680GET ANALYSIS DEVICE* 6GOSUB 1680PRINTPRINT USING 1320

16

1420143014401450146014701480149015001510152015301540155015601570158015901600161016201630640165016601670168016901700171017201730174017501760177017801790180018101820183018401850186018701880189019001910192019301940195019601970198019902000201020202030204020502060207020802090210021102120213021402150

H.-J. Stan and H. Goebel BASIC program to combine data from two selective detectors

PRINTNEXT N1REMREM ********** FOOT NOTES TO THE FINAL REPORTREMPRINT TAB(10); ", BELONGS TO A CRITICAL PESTICIDE THAT INDICATES"PRINT TAB(13); "SIMILAR RETENTION TIME TO A COMPOUND OF ANOTHER"PRINT TAB (27); "STANDARD MIXTURE"PRINTPRINT TAB(10); "+ BELONGS TO A COMPOUND RESPONDING TO ECD AND NPD"PRINTGET REPORT 4 DEVICE* 6STOPREMREM ********** FINAL EVALUATIONSREMFOR J= TO *PEAKS(2)LET Y$ NAMES(J,2)FOR TO *PEAKSIF NAMES(I)= Y$ THEN 1660IF NAME$(J,2)="ALDRIN" THEN 1660IF AMT (J,2) < 0.01 THEN 1660NEXTPRINT NAMES(J,2),"IS IDENTIFIED WITH ECD IN";IDSNEXT JGOTO 1780FOR TO *PEAKSLET X$ NAMES(I)FOR J TO *PEAKS(2)IF NAMES(J,2)= X$ THEN 1770IF NAMES(I)= "PT" THEN 1770IF NAMES(I)= "NT" THEN 1770IF AMT(I)<0.01 THEN 1770NEXT JPRINT NAMES(I), "IS IDENTIFIED WITH NPD IN";ID$NEXTFOR I- TO *PEAKSLET US NAMES(I)LET F AMT(I)FOR J= TO *PEAKS(2)IF NAMES(J,2)= U$ THEN 1840GOTO 1960IF AMT(J,2)> =0.01 THEN 1860GOTO 1960LET E AMT(J,2)IF F > E THEN 1890IF E> =F THEN 1910LET Y-(F-E),100/FGOTO 1920LET Y (E-F), 100/EIF Y < 30 THEN 1950GOTO 1970PRINTPRINT NAMES(J,2), "IS SUSPECTED IN’;ID$NEXT JNEXTRETURNREMREM ********** PRINTING CALIBRATION REPORTS OF STANDARD MIXTURESREM ********** INCLUDING RELATIVE RETENTION TIMESREMFOR TO *PEAKSIF NAMES(I)="NT" THEN 2060GOTO 2070LET A= RT(I)NEXTPRINT HEADSPRINTPRINT "CAL", "NAME", "PPM", "RT", "REL.RT"PRINTFOR TO *PEAKSPRINT CAL*(I),NAME$(I),AMT(I),RT(I),RT(I)/ANEXTRETURN

Figure 1. The program for the NPD channel (leading program).

17

H.-J. Stan and H. Goebel BASIC program to combine data from two selective detectors

LIST PRGMPROGRAM:

1020304050607080901001101201301401501601701801902OO21022023024025O26027028O29030031032033034035O36037038O3904OO41042043044045O4604704804905OO51052O53O54055O56O57058O59O6O061062063064065O66067068O69070O71072073074075O76077078O790

(ANNOTATION OFF)PRINT "PROGRAM (CHANNEL 2). FOR RESIDUE ANALYSIS"PRINT "PARALLEL IN ECD AND NPD"REMREM ********** NPD (SIGNAL A): CHANNELREM ********** ECD (SIGNAL D): CHANNEL 2PRINTINPUT "HOW MANY BOTTLES ARE IN THE TRAY?",NSTARTRECALIBRECALIB RUN TIMEGOSUB 1070STARTRECALIBRECALIB RUN TIMEGOSUB 1070STARTRECALIBRECALIB RUN TIMEGOSUB 1070REMREM ********** INJECTION OF SAMPLES AND STORING THE DATA ON TAPEREMFOR B1 104 TO N+ 100ATTN 2T9THRESHOLD 9STARTLIST CLOCK TIMEPRINT SAMPLE$,ID$PRINT "SIGNAL D"EXECUTE E, "SAVE REPORT "&VALS(B1)&" DEVICE 16"NEXT BPRINTPRINTPRINT TAB(15); "REGISTER OF PESTICIDE RESIDUE OF"; N-3; "SAMPLES"PRINTPRINTIMAGE X,2A,4X,12A,4X,12A,4X,DD.DD,4X,DD.DD,4X,DD.DD,4X,DDDDDDIMAGE X,2A,4X,12A,4X, 12A,4X,5A,4X,5A,4X,6A,4X,6APRINT USING 380; "NO", "SAMPLE", "NAME", "PPM", "RT", "EXP.RT", "AREA"PRINTPRINTFOR BI= 104 TO N+ 100PRINTPRINTPRINT "CALCULATED FROM STANDARD MIXTURE FOR P-COMPOUNDS"REMREM ********** LOADING THE REPORTS INTO MEMORY AND COMPARISON WITHREM ********** THREE STANDARD MIXTURESREMEXECUTE E, "GET REPORT "&VAL$(B1)&" DEVICE 16"REMREM ********** WAITING LOOP FOR SYNCHRONISATIONREM ********** WITH THE MAIN CHANNELREMLET W$ SAMPLES(2)IF W$=SAMPLE$(1) THEN 590WAIT 0.2GOTO 550GOSUB 870REMREM ********** WAITING LOOP FOR SYNCHRONISATIONREM ********** WITH THE MAIN CHANNELREMLET AS HEADSIF AS-"STANDARD MIXTURE II IN ECD" THEN 680WAIT 0.2GOTO 640PRINTPRINT "CALCULATED FROM STANDARD MIXTURE II FOR P-COMPOUNDS"GOSUB 870REMREM ********** WAITING LOOP FOR SYNCHRONISATIONREM ********** WITH THE MAIN CHANNELREMLET B$ HEADSIF B$--"STANDARD MIXTURE FOR CHLORINATED COMPOUNDS IN ECD" THEN 790WAIT 0.2GOTO 750PRINT

18

80081082083084085086087088089090091092093094095096997098099010001010102010301040105010601070108010901100111011201130114011501160117011801190

H.-J. Stan and H. Goebel BASIC program to combine data from two selective detectors

PRINT "CALCULATED FROM STANDARD MIXTURE FOR CHLORINATED COMPOUNDS"GOSUB 870GOTO 930REMREM ********** PRINTING REPORTS AND COMPARISON OF RESULTSREM ********** BETWEEN ECD AND NPDREMFOR I-1 TO *PEAKSIF AMT(I)> =0,01 THEN 900GOTO 910PRINT USING 370; SAMPLES, IDS, NAMES(I), AMT(I), RT(I), EXPRT(I), AREA(I)NEXTRETURNWAIT 0.2REMREM ********** WAITING LOOP FOR SYNCHRONISATIONREM ********** WITH THE MAIN CHANNELREMLET V$ SAMPLES(2)IF V$< >SAMPLES(l) THEN 1010GOTO 930NEXT B1STOPREMREM ********** PRINTING CALIBRATION REPORTS OF THE STANDARD MIXTURESREM ********** INCLUDING RELATIVE RETENTION TIMESREMFOR TO *PEAKSIF NAMES(I)="ALDRIN" THEN 1100GOTO 1110LET A RT(I)NEXTPRINT HEADSPRINTPRINT "CAL", "NAME", "PPM’, "RT’, "REL.RT"PRINTFOR TO *PEAKSPRINT *CAL (I),NAME$(I),AMT(I),RT(I),RT(I)/ANEXTRETURN

Figure 2. The program for the ECD channel.

REGISTER OF PESTICIDE RESIDUE OF SAMPLES

NO SAMPLE NAME PPM RT EXP.RT

CALCULATED FROM STANDARD MIXTURE FOR P-COMPOUNDS4 PEACHES PHOSPHAMI + .544 PEACHES PARATH-ME+ 1.484 PEACHES MALATHION+ 2.694 PEACHES ALDRIN 2.004 PEACHES PARATHION+ .20

AREA

CALCULATED FROM STANDARD MIXTURE II FOR P-COMPOUNDS4 PEACHES DICHLOFENT+ 3.374 PEACHES PARAOXON+ .594 PEACHES ALDRIN 2.004 PEACHES DURSBAN+ * .10

13.97 13.86 176314.21 14.20 4993216.49 16.57 1671616.85 16.85 10099717.21 17.14 2577

14.21 14.18 4993214.98 15.05 560716.85 16.85 10099717.21 17.18 2577

7.6014.9716.85

CALCULATED FROM STANDARD MIXTURE FOR CHLORINATED COMPOUNDS4 PEACHES CHLORFPROP-M .79 7.674 PEACHES HEPTACHLOR, .30 14.984 PEACHES ALDRIN 2.00 16.85

27195607

100997

Figure 3. Print-out from the ECD channel for a screenin9 run of pesticides in peaches.

19

H.-J. Stan and H. Goebel BASIC program to combine data from two selective detectors

REGISTER OF PESTICIDE RESIDUES OF SAMPLES

NO SAMPLE NAME PPM RT

CALCULATED FROM STANDARD MIXTURE FOR P-COMPOUNDS4 PEACHES PARATH-ME+ 1.604 PEACHES PARATHION+ .104 PEACHES NT 2.00

THE DIFFERENCE BETWEEN THE DETECTORS AMOUNTS TO 8%FOR PARATH-ME+THE DIFFERENCE BETWEEN THE DETECTORS AMOUNTS TO 52FOR PARATHION+

CALCULATED FROM STANDARD MIXTURE II FOR P-COMPOUNDS4 PEACHES DICHLOFENT+ 5.114 PEACHES DURSBAN+* .074 PEACHES NT 2.00

EXP.RT AREA

14.20 14.32 108817.20 17.28 2718.19 18.19 1055

14.20 14.28 108817.20 17.31 2718.19 18.19 1055

THE DIFFERENCE BETWEEN THE DETECTORS AMOUNTS TO 34%FOR DICHLOFENT+THE DIFFERENCE BETWEEN THE DETECTORS AMOUNTS TO 35%FOR DURSBAN+

CALCULATED FROM STANDARD MIXTURE FOR N-COMPOUNDS4 PEACHES NT 2.00 18.19 18.19 1055

CHLORFPROP-M IS IDENTIFIED WITH ECD IN PEACHESHEPTACHLOR, IS IDENTIFIED WITH ECD IN PEACHESPARATH-ME+ IS SUSPECTED IN PEACHES

* BELONGS TO A CRITICAL PESTICIDE THAT INDICATES SIMILAR RETENTION TIME TO A COMPOUND OFANOTHER STANDARD MIXTURE

+ BELONGS TO A COMPOUND RESPONDING TO ECD AND NPD

Figure 4. Print-out from the NPD channel for the same screenin9 run as in figure 3, includin9 final report.

Discussion

Figures 3 and 4 show the parallel print-outs from the twoterminals reporting a screening run for pesticide residues inpeaches. By means of this example, the utility and the limits ofour program for evaluating food samples are briefly discussed.

After the clean-up, a number ofsubstances responding to theECD are generally found in a chromatogram and several arerecognized as calibrated pesticides in the screening run. Infigure 3 a total of nine pesticides are indicated, together with theinternal standard (aldrin). Only these nine compounds out ofabout 80 pesticides incorporated in three calibration mixturesmay be present in the sample. Processing of the NPD signalsrecorded simultaneously in the other channel results in fourorganophosphorous pesticides and no nitrogen-containingpesticide(figure 4). For all ofthe compounds responding to bothdetectors a calculation of the response ratios and comparisonwith their calibrated values leads to the discrimination of’parathion’, dichlofenthion’ and ’dursban’, whereas the responseratio of ’parathion methyl’ is close to the calibrated one.Therefore parathion methyl is announced in the final ?eport assuspected. This screening run permits no further decision about’chlorfenprop methyl’ and ’heptachlor’.

All pesticides marked with a cross in figure 3 are organo-phosphorous compounds and so they also have to respond to

the NPD. As there are no corresponding signals for ’phos-phamidon’, ’malathion’ and ’paraoxon’ in the NPD report ofthis example, all three substances are eliminated and do notappear in the final report. This discrimination procedure resultsin the final proposal of chlorfenprop methyl, heptachlor andparathion methyl as possibly being present in this sample.

The program proved to be a great help in routine analysis forselecting the positive food samples after screening. A majordrawback is the time-consuming data transfer between the tapeand the gas chromatograph’s memory.

The analyst has to inspect the chromatograms of bothdetectors in order to evaluate the quality of the separation andthe performance of the chromatographic system. After this, thecomputer supports him by handling the huge amount ofinformation produced by a series of screening runs.

References

1. GOEBEL, H. and STAN, H.-J. in J. Rijks (ed.), Proceedings of theFifth International Symposium on Capillary Chromatography(Elsevier, Amsterdam, 1983), 557.

2. STAN, H.-J. and GOEBEL, H., Journal of Chromatography(in press).

20

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