Anal. Chem. 1983, 55, 994-998
994
Simultaneous Liquid Chromatographic Determination of Antidepressant Drugs in Human Plasma Shude Yang and Merle A. Evenson* Toxicology Laboratory, University Hospital, Departments of Medlclne -Pathology 600 Highland A venue, Madison, Wisconsin 53792
and Laboratory Medicine, University of Wisconsin,
A unique solvent extractlon and a liquid chromatographic assay method have been developed for elght commonly used tricyclic antidepressant drugs in plasma uslng a Zorbax Cyano propylsllane column wlth an unusual moblle phase. The objective of these studles was to develop an analytical method that would utlllre a single solveiit extraction step and an lsocratlc liquld chromatography program that could be used In routlne servlce laboratories and that would be adequately accurate and precise for therapeutic monltorlng of these drugs. The chloroform-ethyl ether solvent extractlon step followed by the frozen separatlon of the plasma extract provldes high yields and a clean sample for the llquld chromatographic assay. Standard addition studies for these drugs and the Internal standard provlded a recovery that ranged from 91 % to 102%. The Ilmlt of detection (LOD) (senslvlty) for each drug in plasma Is 5 ng/mL and the method Is linear to 500 ng/mL. The preclslon studles for both wlthln assay ( n = 12) and between assay ( n = 20) produced coefflclent of varlatlon values of less than 4 % . About 80 bask or neutral drugs were tested for posslble Interference, and only a few Interfere In minor ways wlth thls method.
We report a reverse-phase HPLC method using an unusual acetic acid-amine modifier mobile phase where the eight commonly prescribed tricyclic antidepressant drugs, amoxa8-OH amoxapine (8-OH AMO), doxepine (DOX), pine (AMO), desmethyldoxepin (DESD), imipramine (IMI), desipramine (DESI), amitriptyline (AMI), and nortriptyline (NOR) are separated with base line resolution within 15 min. The detection is at 254 nm and either peak height or peak area ratios of drug to internal standard are used for quantitation. Thus, these eight tricyclic drugs and metabolites can be assayed in a single procedure that is essentially free from interferences. We also developed a very simple, rapid, and effective sample pretreatment solvent extraction procedure. The average absolute recoveries of tricyclic antidepressant drugs and internal standard (7-OH loxapine) carried through the whole assay are above 95% with the exception of 8-OH AMO, which has a recovery of 91%. The combination of the HPLC method and the sample preparation allows us to establish a very precise, accurate, sensitive, relatively inexpensive, and simple method for simultaneous assay of eight tricyclic antidepressant drugs and metabolites in plasma. The whole procedure is ideally suited to a routine, high volume, service and analytical laboratory.
Therapeutic drug monitoring of tricyclic antidepressant drugs for clinical chemistry laboratories is a challenging task. Many contemporary analytical methodologies have been utilized to assay these drugs and critical reviews of the methodologies have been published (1, 2). No one is completely satisfied with these methods in respect to specificity, sensitivity, simplicity, short analysis time, and cost of conducting the measurements. For routine clinical applications, high-pressure liquid chromatography (HPLC) seems to be the most attractive analytical approach. It is convenient and simple and can be relatively inexpensive. However, as with other organic amine compounds, the separation of tricyclic antidepressant drugs by HPLC has usually encountered some difficulties (3-10,13). Poor resolution and tailing peaks are the most common and significant chromatographic problems. In addition the sensitivity for most published methods is too low to monitor therapeutic or subtherapeutic concentrations of tricyclic drugs in plasma using the UV detector at 254 nm. Sonsalla et al. (8) present a HPLC method but it requires silanized glassware and careful attention to the evaporation step and does not include the newer tricyclic drug amoxapine or its active metabolite 8-OH amoxapine. Koteek et al. (9) report a HPLC method but the recovery of the drugs was only 20-44% with solvent extraction and only 72-97% with solid-phase chromatography as the sample preparation method. Kabra et al. (10) report a reversed-phase HPLC method that can separate six tricyclic drugs and metabolites. However, their method does not include amoxapine and 8-OH amoxapine. In addition, a 200-nm UV detector must be used to obtain the necessary sensitivity, special glassware preparation is still required, and the recovery of amitriptyline is only about 70%.
Apparatus. A Model 6000 A delivery system, Model 440 UV detector (254 nm), WISP Model 710-B autoinjector, Model 730
EXPERIMENTAL SECTION
0003-2700/83/0355-0994$0 1.50/0
Data Module, and Model 720 system controller (all from Waters Associates, Milford, MA) were used with a 4.6 mm X 25 cm, 6-8 fim particle size Zorbax Cyanopropylsilane (CN)solumn (Du Pont, Wilmington,DE). A 5-cm guard column packed with Permaphase ETH (Du Pont, Wilmington, DE) is used to protect the analytical column. Reagents. All reagents and chemicals were reagent grade or better. A list of some of the special reagents follows: Acetonitrile, methanol, and chloroform (HPLC grade) were obtained from Fisher Scientific Co., Fair Lawn, NJ. The glacial acetic acid (HPLC grade) was from the J. T. Baker Chemical Co., Phillipsburg, NJ. The hexane (distilled in glass) was from Burdick and Jackson Laboratories Inc., Muskegan, MI. The ethyl ether anhydrous (A.R.) was from Mallinckrodt, Inc., Paris, KY. The n-butylamine (99%) was from Aldrich Chemical Co., Inc., Milwaukee, WI. The saturated borate buffer, pH 11,was prepared by adding enough 6 mol/L NaOH to the saturated sodium borate solution t o bring it to a pH 11. Mobile Phase. The mobile phase is prepared by mixing together 540 mL of 0.5 mol/L acetic acid solution and 460 mL of 0.03% (v/v) n-butylamine in acetonitrile. The mobile phase is then degassed with helium or filter-degassed through a Millipore system (Type FS, 3.0 Fm, Millipore, Bedford, MA). Extraction Soluent. The extraction solution is a ethyl ether/chloroform mixture (80/20) (v/v). It is freshly prepared each month and is kept well sealed to prevent evaporation. Drugs. The drugs, as their hydrochloride salts, were obtained as gifts from the following sources: AMI (Merck Sharp and Dohme Research Lab, West Point, PA), NOR (Eli Lilly Co , Indianapolis, IN), IMI (Geigy Pharmaceuticals, Ardsley, NY), DESI (Lakeside Labs, Milwaukee, WI), DOX and DESD (Pfizer Inc., Groton, CT). AMO, 8 - O H AMO, and 7-OH loxapine as free bases were from Lederle Laboratories, American Cyanamide Co. (Pearl River, NY). 0 1983 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
1
095
Tricyclics method
I ,
0
0
40
80
120
160
200
Time (min) Figure 1. Chromatogram of plasma pool containing each of the tricyclic drugs at 150 n g h L and the internal standard at 200 ng/mL. Standards. Stock Drug Standard. Each drug is dissolved in methanol to prepare stock standards containing 0.1 mg/mL of drug. This solution is stable in the freezer in the dark for at least 1 year. Composite Drug Standard. Mix together 1.0 mL stock standard of each drug and add 2 drops of 0.1 mol/L HC1 to maintain the drugs in the hydrochloride salt form. Next add 10.0 mL of methanol and then dilute to volume with distilled water in a 100-mL volumetric flask. Store in freezer. This standard contains 1.0 Fg/mL of each drug. Plasma Standard. Working plasma and controls are made by adding various amounts of composite standard into drug free plasma. Internal Standard. Stock standard is prepared by adding 10 mg of 7-OH loxepine to a 100-mL volumetric flask and diluting to the mark with methanol. Take 2.0 mL of this stock standard into a 50-mL volumetric flask, add 2 drops of 0.1 mol/L HCl, dilute to mark with distilled water, and store in a freezer. This final internal working standard contains 4 pg/mL of 7-OH loxapine. Procedure. Transfer 2.0 mL of serum oir plasma into a 12-mL glass centrifuge tube, add 100 fiL of the 4 pg/mL internal standard, 7-OH loxapine, and 1.5 mL of pH 11 saturated borate buffer, vortex-mix for a few seconds. Add 6 mL of extraction solvent, stopper the tube tightly, and manually shake it for 8 min. Centrifuge for 5 min at 300Og. Put the tube into a methanol-dlry ice bath for a few minutes, pour off the upper organic layer into another centrifuge tube and evaporate the organic contents j u s t to dryness at ambient temperature, using a stream of nitrogen or air to aid evaporation. The tubes must not be allowed to be completely dry under the stream of air or nitrogen for more than a few seconds otherwise losses will occur. Add 0.2 mL of mobile phase and 0.2 mL, of hexane to the dry extract tube and vortex-mix for a few seconds and thlan centrifuge for 5 min. After discarding the top hexane layer, inject 40 WLof the reconstituted mobile phase into the chromatograph under the following chromatographic conditions: the imobile phase flow rate is at 2.5 mL/min; the UV detector is at 254 nm, the sensitivity is at 0.005 A full scale (AUFS), and the temperature is at ambient. No specific glassware preparation is needed. Each drug was quantitated by measuring the ratio of the peak height of each drug to that of internal standard in the unknown sample and comparing it with a plasma standard of known concentration.
RESULTS Figure 1illustrates a typical chromatogram of the working standard mixture of ithe tricyclic drugs and the internal standard added to the dlrug free plasma pool. Notice that base
40
80
120
160
200
Time ( m i n ) Figure 2. Chromatogram of tricyclic free plasma sample.
line resolution is obtained. Comparison of the blank plasma t o the reagent blank produced no peaks with retention times near any of the drugs. A chromatogram of the plasma pool sample, known to be free of tricyclic drugs, is shown in Figure 2. For the absolute recovery determinations, known amounts of drugs and internal standard were added to drug-free plasma and then carried through the extraction process. These values were then compared directly to standards that were not extracted. Comparison of peak heights from extracted samples with the peak heights from standards was then used for calculating the absolute analytical recovery. The recoveries of eight tricyclic drugs and internal standards were measured at three diffrent concentrations, ranging from 40 to 1000 ng/mL. As indicated in Table I, extraction efficiencies are nearly quantitative a t each concentration for all drugs. The linearity and sensitivity calibration curves were obtained by plotting the ratio of the peak heights of each of the tricyclic drugs t o that of the internal standard. The concentrations in control plasma are linear from 25 to 500 ng/mL and pass through the origin. Regression analysis data for individual drugs are given in Table 11. The within-day and day-to-day precision values were established with spiked pools of plasma a t two different concentrations, corresponding roughly to the lower and upper limit of the therapeutic range reported for most tricyclic drugs. The results are listed in Table 111. Eighty commonly prescribed basic and neutral drugs that might be partially extracted were checked for possible interference. Most of the drugs are eluted earlier than the retention time of the tricyclic drugs. Only ten drugs listed in Table IV were eluted within the range in retention time of tricyclic drugs. At usual therapeutic concentrations in serum, pheniramine, maprotiline, and pyrimethamine do not interfer with tricyclic drugs due to their low sensitivities a t the 254-nm detector. For practical purposes, it will be very rare for a patient sample t o contain all of the eight tricyclic drugs a t the same time; therefore if an interfering drug is encountered when quantitating a particular tricyclic drug, it is possible to change the ratio of mobile phase, and change the resolution enough to avoid such interference. The drugs that were found not to interfere and that eluted much earlier than the tricyclic drugs are acetaminophen, acetaphenetidine, amobarbital, barbital, bendroflumethiazide, benadryl, benthiazide, benztropine, butalbital, caffeine, carbamazepine, carisoprodol, chlordiazepoxide, chlorpropamide,
996
ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
Table I. Recovery of Tricyclic Drugs and Internal Standard from Plasma
drug 8-OH AM0
AM0
DESD
DOX
DESI
NOR
IMI
AMI
7-OH loxapine (1.S.)
amt added, ng/mL 38.0 38.0 190 190 950 950 41.0
41.0 205 205 1025 1025 42.2 42.2 212 212 1060 1060 42.2 42.2 212 212 1060 1060 41.2 41.2 206 206 1030 1030 35.4 35.4 177 177 885 885 42.8 42.8 214 214 1070 1070 40.2 40.2 201 201 1005 1005 36.8 36.8 184 184 920 920
amt recovered, ng/ mL 33.8 33.1 179 175 874 893 38.1 38.5 203 203 984 953 42.2 43.9 210 212 1102 1091 41.4 43.9 212 212 1081 1060 41.2 43.3 2 04 204 1040 1020 36.5 37.9 174 170 911 885 44.9 45.4 214 210 1059 1047 39.8 41.0 201 201 985 985 34.2 35.3 182 182 874 883
Table 11. Linearity of Peak Height Ratio ( Y )with Concentration (X)in Control Plasma (25-500 ng/mL)a
recovery, o/c average 89 87 94 92 92 94 93 94 99 99 96 93 100
91.3
95.7
drug
corr coeff
Y intercept
slope x lo3
8-OH AM0 AM0 DESD DOX DESI NOR IMI AMI
0.9994 0.9977 0.9987 0.9985 0.9993 0.9991 0.9986 0.9990
-0.015 -0.009 -0.006
6.287 5.186 5.962 4.590 5.007 3.311 3.931 2.883
101.7 drug 8-OH AM0 100.7
AM0
100 100
DESD
102
DOX 100.5
105 99 99
IMI 101.2
101.0
100
98 99 98 99 102
99.5
100 100
98 98 93 96 99 99 95 96
AMI
within day ( n = 12) day to day ( n = 20) corr corr range, ng/mL coeff, range, ng/mL coeff, ( i s t d dev) % (*std dev) % 47.4 * 1.5 237 i 5.45 51.2 i. 0.92 256 i 4.4 53.0 f 1.5 265 I 8.2 53.0 i 1.4 265 i 5.0 51.5 f 1.5 258 i 8.2 44.3 * 1.3 222 i 6.9 53.6 f 1.9 268 i. 7.5 50.2 i 1.8 251 * 5.8
3.1 49.6 f 1.7 2.3 262 i 9.3 1.8 5.3 f 1.8 1.7 267 i 8.7 2.8 55.7 f 1.8 3.1 274 f 8.9 2.6 56.1 t 2.0 1.9 278 t 8.2 3.0 53.8 i 1.9 3.2 267 i: 6.3 3.0 46.8 f 1.8 3.1 230 i. 7.7 3.6 56.8 i 2.1 2.8 282 -L 10.4 3.6 52.3 * 1.6 2.3 263 i 10.0
3.4 3.6 3.4 3.3 3.2 3.3 3.6 3.0 3.5 2.4 3.8 3.4 3.6 3.7 3.0 3.6
Table IV. Relative Retention Time (RRT) of Some Drugs
100
105 106
DESI NOR
101
99 103 107 98 96 103
+ 0.006
Table 111. Precision of the Assay
100
100 100
t 0.004
* Calculations based on 12 woints for each drug.
104 99 104 103 98 104
0.000
-0.005 -0.003
96.3
chlorthalidone, clonazepam, propoxyphene, demoxepam, ndesalkylflurazepam, n-desmethylchlordiazepoxide, ndesmethyldiazepam, diazepam, 10,ll-dihydrocarbamazepine, phenytoin, diphenoxylate hydrochloride, 4-hydroxypropranolol, doxylamine, glutethimide, flunitrazepam, furosemide, hydroflemthiazide, lorazepam, meperidine, meprobamate, mesantoin, methaqualone, primidone, nirvanol, nitrazepam, noludar, norvalium, oxazepam, penicillamine, pentobarbital, phenobarbital, phenylbutazone, polythiazide, propranolol, prazepam, salicylamaide, salicylate, secobarbitol, scopolamine, tolbutamide, temazepam, theobromine, and theophylline.
drug
RRT
note
8-OH AM0 pyrimethamine 7-OH loxapine pheniramine AM0 DESD loxapine methapyridene DOX pyribenzamine DESI sparine desmethylcyclobenzapine protriptyline maprotiline NOR IMI cyclobenzaprine AMI
0.83 0.97
low sensitivity
1.00 1.01
low sensitivity
1.30 1.45 1.52 1.52 1.61 1.70 1.72 1.76 1.77 1.78
1.80 1.83 1.93 1.97 2.05
low sensitivity
The drugs that were found not to interfere and the elute much later than the tricyclic drugs are chlorpromazine, clomidpramine, mellaril, fluphenzine, prochlorperazine, and trifluoperazine. If these drugs were present and appeared on the chromatogram of the next sample, it would be obvious due to the wide peak width. Because the drug was on the column so long, the diffusion of the band would cause such a broad peak that it would be obvious the compound was from the previous injection. Method comparison studies between GLC and HPLC for all eight drugs were conducted on the same patient sample
ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983 * 997
usually analyzed on the same day. For NOR for n = 16 the mean ( f ) of the GLC! method was 108 ng/mL, the f of the HPLC methodl was 911 ng/mL, and the correlation coefficient (R)was 0.9704. For AMI for n = 13 the f of the GLC method was 146 ng/mL, the 3; of the HPLC method was 161 ng/mL and the R was 0.9397. For DESIP for n = 9 the x of the GLC method was 122 ng/mL, the R for the HPLC method was 124 ng/mL, and the R was 0.9233. For IMI[ for n = 8 the f of the GLC method was 78 ng/mL and for the HPLC the f was 84 ng/mL and thLe R was 0.9633. The numbers of comparison tests for DOX, DDOX, AMO, and 8-OlH AM0 were too few to do statistical analysis but no bias was observed by inspection of the raw data for these drugs. On the basis of composite values of the literature and our experience, we use the following expected therapeutic ranges: amitriptyline, 80-250 ng/mL; nortriptyline, 50-150 ng/mL; the sum of amoxapine plus 8-OH amoxapine, 200-500 ng/mL; doxepin, 75-250 ng/niL; desmethyldoxepin, 110-250 ng/mL, impramine, 150-250 ng/mL; and desimpramine, 150-300 ng/mL. Several lots of drug free plasma and patient samples have been checked stochastically for potential endogenous interferences. No extraneous peaks are detected except sometimes an unknown peak with a retention time of 3.95 was found. However, it is well separated from 8-OH AMO, the adjacent tricyclic drug and will usually not cause an analytical problem. The comparisons of patient’s samples analyzed by our HPLC method and the gas chromatographic method now used in our service laboratory are usually excellent. However, for nortriptyline, GLC data are sometimes higher than the HPLC method. We have found our GLC method for tricyclic drugs involving two extractions and derivative formation prior to detection with the N-1’ detector to be more likely to encounter interferences jhom other drugs and endogenous substances than this HPLC method. We conclude that this HPLC method is significantly more accurate for these drugs in plasma.
DISCUSSION Different types of reverse-phase and adsorption columns with different chemically bonded phaees were tested to separate these eight tricyclic drugs. The separation of these drugs was improved lby adding an organic amine to the mobile phase almost in all cases. The function of tlhe organic amine as a modifier in the mobile phase may not be only ion suppression, particularly in a relatively acidic mobile phase as used with this method. At low pH, modifier and tricyclic drugs will exist primarily in the ioniized form (the pK, values of tricyclic antidepressant drugs are around 9.5). Thus, the main function of an amine in mobile phase probably is competing with the tricyclic drugs (also amines) for a site on the bonded-phase surface. On tlhe basis of on this assumption, we imagine it is possible to improve some of other difficult separation problems of other kinds of compounds bly adding a “competing modifier” into the mobile phase. The competing modifier is similar in chemical structure and chemical property to those that need to be separated. If this approach could be adapted to other kinds of compounds, it would undoubtedly, as in this case, make reverse-phase chromatography on an adsorption type column more effective and powerful. Establishment of the proper p H was found to be quite important to be able to achieve the type of efficiency needed for this application. The mobile phase we used is a mixture of 540 mL of 0.5 moll/L HAC and 460 mL of CH&H containing 0.03% n-butylamine. No external buffer was added to this system. However, this system still has some of the buffering capacity, blecause some conjugate base OAC- will be formed according to the following equation: RHNz
+ HOAC
-+
RNHS+
+ OAC-
Accurate measurement of the pH value of this mobile phase with general pH electrodes is not appropriate, since there is a large amount of organic solvent in this system. According to the calculation and rough pH measurement, with pH paper and a glass pH electrode, its “pH” is around 3.5. A more concentrated pure acetic acid ( 5 % ) solution as mobile phase has been reported in reverse-phase chromatography using p-Bondapak/CI8 column and apparently has not caused damage to the column packing materials (11). Initially, when chemically bonded liquid-phase chromatography columns became commercially available, it was anticipated that presaturation between mobile phase and stationary phase would not be required (12).More recently many researchers and most companies now recommend that guard columns and silica saturating precolumns be used. Our mobile phase is quite different from the usually used one in HPLC and only slight column deterioration was observed early in the study if the mobile phase was not presaturated with the “stationary phase” and silica. In spite of this, our solution to this minor problem was simple and was effective. We simply recycled the mobile phase and we were able to use the same 900-mL mobile phase in recycling mode for 4 months. We have not found any noticeable change in the absorbance background, in the performance of the single column or its separations on over 500 samples during that time. However, minor adjustments to “zero” the background are made each day. When the column does change slightly, the concentration of n-butylamine is increased from 0.03% to 0.035% in the CH&N and the resolution and retention time return to that of a new column. Moreover, there are some other advantages when the mobile phase is recycled. One saves solvent, reduces reagent preparation time, and simplifies the analytical procedures. Since, the column is stored in the mobile phase at all times, washing the column with other organic solvents for conditioning and equilibration of the column in preparation for analytical tasks is not necessary. Solid-phase extraction procedures using Bond-Elut 18 (Analytichem International Inc., Harbor City, CA) were initially applied and resulted in nearly quantitative recoveries under appropriate conditions using CHC13/2-propanol (9/ 1, containing 4% (v/v) n-octylamine) as the final elution solvent. For routine clinical assays in service laboratories, we believe that liquid-liquid extraction is simpler and more convenient. Hence, a very efficient liquid-liquid extraction procedure was developed. The extraction difficulty we first encountered was that these eight tricyclic drugs and the internal standard have a wide range of polarity. The solvents reported in the literature for extracting tricyclic drugs resulted in low recovery for A M 0 and particularly for 8-OH AMO. We believe this observation occurred because of the polarity of these drugs. The solvent extraction procedure described here solved this recovery problem. To simplify the procedure for separating two liquid phases after extraction, we used a dry ice bath to freeze the aqueous phase and then pour off the upper organic solvent into another tube. Hence another condition that the extracting solvent system had to meet was the specific gravity of the solvent mixture must be less than that of the aqueous phase. In addition, the extraction solvent should also have a low boiling point for easy evaporation. We found that with extraction a t pH 11 (saturated borate buffer) with ethyl ether/chloroform (80/20 (v/v)), we can meet all the above requirements. We did not find any noticeable loss of drug due to absorption on glassware in our method; thus, special glassware preparation is not necessary. There may be two reasons for observing this phenomenon. During extraction, the surface of glassware contacts a large amount of substances ranging from low-polar volatile solvent to very polar compounds from
998
Anal. Chem. 1983, 55, 998-1001
plasma and most of the active sites on the glass are perhaps covered. Consequently, there is low probability for small amounts of tricyclic drugs to be selectively adsorbed on the glassware. During the second step, the residue obtained after evaporating organic solvent is reconstituted with mobile phase containing n-butylamine. The amine concentration is at least 1 order of magnitude higher than that of tricyclic drugs, and perhaps the amine modifier saturates the active sites on the glass. These conditions then allow for nearly quantitative recovery of all drugs, which other methods have not achieved. In the drying process, some of the organic amines are more susceptible to degradation in free base form than in their salt form. Therefore, we do not suggest that the extracts remain at dryness for more than a few minutes after the organic solvent has been evaporated. We used an air or nitrogen stream to aid evaporation, and we have not found drug loss at the room temperature evaporation.
ACKNOWLEDGMENT We thank all of the drug manufacturers who provided the pure drugs as gifts. We also thank the People’s Republic of China for the opportunity of Shude Yang, a visiting scholar on leave from the Chinese Academy of Medical Sciences, Peking, China, to come to the University of Wisconsin, Madison. Registry No. Amoxapine, 14028-44-5;8-hydroxyamoxapine,
61443-78-5; doxepin, 1668-19-5; desmethyldoxepin, 1225-56-5; imipramine, 50-49-7;desipramine,50-47-5;amitriptyline, 50-48-6; nortriptyline, 72-69-5.
LITERATURE CITED (1) Scogglns, B. A.; Maguire, K. P.; Norman, T. R.; Burrows, G. D. Ciin. Cbem. (Winston-Salem, N . C . ) 1980, 2 6 , 5-17. (2) Gapta, R. N.; Milnar, G. Biopharm. Drug Dispos. 1980, I , 259-278. (3) Vandernark, F. L.; Adarns, R. F.; Schrnldt, G. J. Clin. Chem. (WinstonSalem, 1978,, 2 4 , 87-91. (4) Wallace, J. E.; Shlrnek, E. L., Jr.; Harris, S. C. J . Anal. Toxicoi. 1981, 5 , 20-23. (5) Proeles, H. F.; Lohman, H. H.; Miles, D. G. Ciin. Cbem. (Winston-Saiem, N.C.)1978, 2 4 , 1948-1953. (6) Thorna, J. J.; Bondo, P. B.; Kozak, C. M. Tber. Drug Monit. 1979, 7 , 335-338. (7) Bannlster, S. J.; VanderWal, S.J.; Dolan, J. W.; Snyder, L. R. Clin. Cbem. (Winston-Salem, N . C . ) 1981, 2 7 , 849-855. (8) Sonsalla, P. J.; Jennison, T. A,; Flnkle, 8. S. Clin. Chem. (WinstonSalem, N . C ) 1982, 28, 457-481. (9) Koteel, P.; Mulllns, R. E.; Gadsden, R. H. Ciin. Cbem. (Winston-Salem, N.C.)1982, 28, 462-466. (10) Kabra, P. M.; Mar, N. A.; Marton, L. J. Ciin. Cbim. Acta 1981, I l l , 123- 132. (1 1) Smyly, D. S.; Woodward, B. B.; Conrad, E. C. J . Assoc. Off. Anal. Cbem. 1976, 59, 14. (12) Snyder, L. R.; Kirkland, J. J. “Introduction to Modern Liquid Chromatography”, 2nd ed.; Wlley: 1979; p 270. (13) Tasset, J. J.; Hasson, F. M. Ciin. Chem. (Winston-Salem, N . C . ) 1982, 28, 2154-2157.
RECEIVEDOctober 5 , 1982. Resubmitted March 2, 1983. Accepted March 2, 1983.
Effects of Interferogram Sampling of Gas Chromatography/Fourier Transform Infrared Data on Gram-Schmidt Chromatogram Reconstruction Robert L. Whlte, Gary
N. Glss, Gregory M. Brlssey, and Charles L.
Wllklns”
Department of Chemistry, University of California -Riverside, Riverside, California 9252 1
A theoretlcal and experlmental study of the effect of changlng Interferometric sampling of gas chromatography/Fourler transform Infrared (GC/FT-I R) Gram-Schmldt reconstructed gas chromatograms (GC) on peak signal-to-noise ( S I N ) rallo and relative peak heights Is presented. I t Is determlned that no generally optimum vector dlsplacernent exists because identity of the GC eluents and instrumental instablllty determlne whlch portion of an Interferogram will produce the greatest GC/FT-IR sensitlvlty.
In 1977, de Haseth and Isenhour reported the first application of the Gram-Schmidt vector orthogonalization procedure to reconstruction of gas chromatography/Fourier transform infrared (GC/FT-IR) chromatograms (1). The major advantage of the technique is that chromatograms can be calculated by using time-domain interferometric data and the time-consuming process of Fourier transformation of each acquired data file can be avoided. Because only a small portion of the interferometric data is used in Gram-Schmidt reconstructions, de Haseth and Isenhour attempted to determine which portion of the raw data produced the best chromatograms. Using data acquired during a single GC/ FT-IR separation, they empirically determined an optimum
vector sampling for 2048-point interferograms to be a region starting 60 data points to the right of the centerburst position and extending right for 100 data points. Subsequently, GC/FT-IR manufacturers provided users with Gram-Schmidt reconstruction software employing this vector sampling. In addition, Isenhour and co-workers have shown that quantitative analysis is possible by using Gram-Schmidt GC/FT-IR reconstructions and that the empirically determined “optimum” interferogram region can be useful for inteferogram-based library searches (2, 3 ) . In 1981, we reported that simplex optimization of four test mixture Gram-Schmidt GC/FT-IR reconstructions resulted in a different optimum vector displacement for each separation when average SIN for the chromatograms was maximized (4). In each instance, the optimum vector sampling included data in the vicinity of the centerburst. This was inconsistent with the earlier report by de Haseth and Isenhour which suggested the optimum sampling was 60 data points away from the centerburst. Comparison of simplex-derived optimum chromatograms with chromatograms generated by using data 60 points away from the centerburst position revealed that relative chromatographic peak heights for mixture component eluents varied when the vector sampling region was changed. These results indicate that a generally applicable optimum vector sampling for Gram-Schmidt reconstructions may not
0003-2700/83/0355-0998$01.50/00 1983 American Chernlcal Soclety
