Semiautomated, Specific Routine Serum Cholesterol Determination by Gas-Liquid Chromatography J. L. Driscoll, D. Aubuchon, M. Descoteaux, and H. F. Martin Pathology Department, Rhode Island Hospital, Providence, R. I . By gas-liquid chromatography, a specific, routine, semiautomatic method for determining serum cholesterol levels at a rate of 40 samples per hour is reported. Using peak height ratio measurements, heptane extracts of saponified serum specimens containing an internal standard are chromatographed with the resulting cholesterol data having an accuracy of better than 97% and a coefficient of variation of the order of 5%. The gas-liquid chromatographic results accumulated over a 12-month period agreed very favorably with cholesterol values obtained by other laboratories using the Abell-Kendall procedure.
WITH THE ADVENT of multiple screening tests, the need for greater accuracy in clinical methods is paramount since false positives can lead to increased cost in diagnostic workup, while false negatives invalidate the objective of the screening test. Included in most screening profiles is the determination of serum cholesterol because of its relationship t o hypertension, hyperlipidemia, and atherosclerosis. However, the literature (1-3) clearly documents the inadequacies of existing methods. I n our laboratory we have made a great effort to introduce specific methods which can be calibrated with the best available primary standards. Implementation of this goal is dependent on the availability of a prime standard and an understanding of the underlying chemical reactions. The latter consideration is imperative in this age of polypharmacy where analytical errors resulting from the presence of drugs are not rare situations (4, 5). I n the case of cholesterol, primary standards are available but the lack of understanding of the chemistry of the various chromophore-producing reactions still exists. In an attempt to build specificity into a method for the determination of cholesterol, we had considered selective bromination, an enzymatic method (6) and gas-liquid chromatography (GLC). Selective bromination should fail because of the presence of unsaturated fatty acids and the enzymatic approach was dismissed because of the lack of the commercial availability of cholesterol dehydrogenase. Consequently, utilization of G L C was the only reasonable alternative. The utilization of G L C for the determination of steroids, lipid components, and drugs in biological fluids is well documented (7-15). However, it has not been extensively used for routine methodology in the clinical laboratory. (1) “Clinical Chemistry Principles and Techniques,” J. R. Henry, Ed., Harper & Row, New York, N. Y., 1964, pp 843-864. (2) D. B. Tonks, Clit7. Biocltem., 1, 12-29 (1967). (3) R. N. Barnett, A. D. Casl, and S. P. Jughans, New Eng. J.
Med., 279, 974-979 (1968). (4) W. A, Wirth and R. L. Thompson, Amer. J. Clin. Path., 43, 579 (1965). (5) F . C. Cross, A. T. Canada, and N. M. Davis, Amer. J. Hosp. Pliarm., 23, 234 (1966). (6) T. C. Stadtman, “Methods in Enzymology,” Vol. 1, Academic Press, New York, N. Y., 1955, pp 678-681. (7) H. H. Wotiz and H. F. Martin, Anal. Biochem., 3, 97 (1962). (8) A. T. James, “Methods of Biochemical Analysis,” Vol. VIII, Interscience, New York, N. Y., 1963, pp 1-59.
1196
In the past, instrumental performance has been poor and operational procedures as well as sample preparation have been too lengthy for handling large work loads. Today, with improved design and construction, there should be n o reason why a G L C unit, assembled with module electronics, easily replaceable heating elements, and injector ports and detectors that are readily cleaned, cannot be considered a reliable instrument. This communication is a report on the development of a semiautomated routine gas chromatographic method for the specific determination of serum cholesterol (total or esters). This method has been successfully used for a year in conjunction with our Multiphasic Screening Center. Sample preparation is minimal and with the use of an automatic injector, the analysis rate is 40 samples per hour. The internal standard technique is used and quantitation was based on the measurement of peak height ratios. Based on the recovery of cholesterol from samples containing known amounts of palmitate and stearate esters, the accuracy was better than 97% and the coefficient of variation of the order of 5 %. The normal ranges associated with this method agree with those established with the Abell-Kendall method (16), the accepted reference method. EXPERIMENTAL
Chemicals and Materials. The following were used : potassium hydroxide, Mallinckrodt AR Grade; ethyl alcohol 95 %, USP grade; heptane, n-octacosane, Eastman White Label Grade; cholesterol, National Bureau of Standards cholesteryl palmitate and stearate and Mann Research (99 99 Applied Science Laboratories; SE-30-silicone gum rubber, Analabs; and Anakron ABS 60170 Mesh, Analabs. Reagents. The potassium hydroxide solution contained 33 grams per 100 ml of solution. The alcoholic potassium hydroxide solution was made by diluting 6.0 ml of the 33 gram solution to 100 ml with ethyl alcohol. The extracting solvent was heptane containing 50 mg of n-octacosane per 100 ml. Cholesterol standards were 50, 75, 100, 125, and 150 mg of cholesterol dissolved per 100 ml of extracting solvent. Equipment. A Varian Aerograph 1740 Series gas chromatograph equipped with dual channel hydrogen flame detectors was used with a Varian Aerograph Model 30 dual channel recorder. The hydrogen generator was an Elhygen
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(9) A. Kuksis, ibid., Vol. XIV, pp 325-54. (10) E. C. Horning and W. J. A. Vanden Heuvel, “Qualitative & Quantitative Aspects of Separation of Steroids,” Vol. 1, Marcel Dekker, New York, N. Y., 1965, pp 153-198. (11) A. Kuksis, D. Stachnyk, and B. J. Holub, J. Lipid Res., 10, 660-667 (1969). (12) C. Crotte, A. Mule, and N. E. Planche, Clirt. Chim. Acta, 27, 331 (1970). (13) H. F. Martin and J. L. Driscoll, ANAL.CHEM.,38, 345 (1966). (14) B. J. Gudzinowicz, “Gas Chromatographic Analysis of Drugs and Pesticides,” Marcel Dekker, New York, N. Y., 1967. (15) “Handbook of Analytical Toxicology,” I. Sunshine, Ed., Chemical Rubber Co., Cleveland, Ohio, 1969, pp 809, 896, 990. (16) L. L. Abell, B. B. Levy, B. B. Brodie, and F. E. Kendall, J. Bid. Chem., 195, 357 (1952).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
Electrolytic Ultra-Pure Hydrogen Generator (Pressure regulated Model). The automatic injector was a Hewlett Packard Automatic Sampler 7670 A. This unit designed for the Hewlett-Packard instruments was mounted o n the Varian unit in the following fashion. A 9l/q X 7 X ' / d n c h plate of mild steel was drilled with four I / , x 20-inch tapped holes o n one end and four 1 x '/&ch slots o n the other end. These exact positions are shown in Figure I . This plate provided a necessary extension of the manufacturer-supplied mounting bracket so that the HewlettPackard unit was properly positioned with the injection port on the right side of the Varian gas chromatograph. The side with the slots was secured t o the sampler chassis and the side with the tapped holes secured to the mounting bracket. The slots o n the mounting bracket and plate provided sufficient flexibility so that the locator bracket o n the injector could be properly aligned. The septum retainer of the Varian was filed flat and the orifice for the syringe bored to l/a-inch i.d. Proper alignment of the sampler was not difficult but could have been aided if a collar for the septum retainer, temporary or permanent, had been made to fit the locator bracket. Design of a n appropriate septum retainer should accomplish the same purpose. Because we could not use the special septum retainer designed by Hewlett-Packard for use with the sampler, an uneven distribution of force affecting the needle alignments occurred along the needle guide when the injection was made. We eliminated this difficulty by securing a 1/8-inch screw and nut in the bottom of the slot of the locator bracket. This screw acted as a stop for the needle guide to press against while injecting. I n order to introduce samples a t the rate of 40 per hour, the Sample Wash and Injection Cycle was set at Minimum, for which the combined time was 60 seconds. The Programmer was set for a 15-second time lapse between cycles. As normally provided, the programmer was calibrated in increments of minutes. At our request, the manufacturer altered the time base of our unit to increments of 15 seconds. This alteration required the change of one circuit board. Column Packing and Columns. Five hundred milligrams of SE-30 were dissolved in 150 ml of toluene. This solution was then repeatedly poured over 10 grams of Anakrom ABS contained in a glass wool stoppered funnel until 80% of the toluene solvent had vaporized. The solid support was then dried under vacuum a t 40 "Cand packed in two-foot, 1/8-inch 0.d. copper columns by gently tapping the tubing. Approximately 0.5 gram of packing was needed per column, and small glass wool plugs were placed in both ends to contain the material. At no time was the column packing exposed to the atmosphere at a n elevated temperature. Gas Chromatographic Condition. The column temperature was 250 "C; injector port temperature, 270 "C;detector temperature, 275 "C; nitrogen carrier gas inlet pressure, 20 psi; hydrogen gas flow rate, 20 ml per minute; compressed air flow rate, 200 ml per minute; and electrometer sensitivity A per millivolt. setting, 16 X Each column was conditioned a t 250 "C for 48 hours prior to use. Two microliters of a solution containing 300 mg of cholesterol per 100 ml of heptane was then repetitively injected (electrometer setting 63 X 10-lo A per mV) until a constant chromatographic pattern was obtained. The retention times for the internal standard and cholesterol from column to column have been consistently similar t o those shown in Figure 2 . I n those instances where the retention times were slightly longer or shorter than desired, altering the carrier gas inlet pressure (plus o r minus 2 psi) from the established setting provided the necessary compensation. Sample Preparation. A 0.500 ml of serum and 5.0 ml of alcoholic potassium hydroxide solutions were added to a 150 X 15 mm test tube and mixed o n a "Super-Mixer"
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Figure 2. Chromatograph of cholesterol standards (Lab-Line Instruments, Melrose Park, Ill.), The saponification was allowed to proceed for 3 hr in a 37 "C bath in the cork-stoppered test tube; 1.00 ml of the heptane extracting solvent was then added and the solution was well mixed; 5 ml of water were added and the solution was mixed again, After the two phases had settled, approximately 0.5 ml of the heptane extract was placed in the sample vials of the automatic sampler. A 2-111 aliquot of each sample was chromatographed. To determine free cholesterol, 0.500 ml of serum was mixed with 5.0 ml of ethanol and then processed as above. Instrumental Calibration and Calculations. Following five injections of the 300 mg %cholesterol standard, five additional standard solutions were injected, which correspond to 100, 150, 200, 250, and 300 mg of cholesterol in sera due to the 1 :2 dilution in sample preparation. Standardization, Figure 2, was run with the first 25 samples in addition to 2 quality control samples, and repeated whenever the 1.5-minute interval between samples was interrupted. The peak height ratio of cholesterol to internal standard, octacosane, was taken as the ordinate and cholesterol concentration as the abscissa. Over the range of calibration, the graphical representation of the data was linear. A least squares treatment of the calibration data programmed on an Olivetti Underwood Programma 101 provided the slope and intercept whenever
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10,AUGUST 1971
1197
necessary. In a second program, the peak height and base line for each peak were entered and with the above constants stored, the cholesterol concentration of the unknown was calculated. DISCUSSION
The references to the gas chromatographic determination of cholesterol and its chemical derivatives are too numerous to cite. However, Cawley et al. (17) in this country and Curtius and his coworkers (18) in Germany, appear to be the only investigators who have attempted to evaluate G L C as a clinical method for cholesterol. Schmit and Mather (19) have reported a method which was rapid but clinical data and information concerning application are not available. These studies demonstrated the accuracy of G L C as well as some of its impractical aspects, which can be minimized or eliminated. For a rapid determination rate, the retention time, rt, for cholesterol must be minimal, and to achieve this goal 2-foot chromatographic columns were used. By utilizing these short columns, the desired chromatographic conditions were established with moderate carrier gas pressure and column temperature settings, optimizing the flame stability of the detector and the life of the column. Although limited resolution was available with these columns, it was adequate for resolving the internal standard (octacosane) and cholesterol as shown in Figure 2, the only components present in significant concentration in the heptane extract. With respective rts of 30 and 66 seconds, on a single channel of the chromatographic unit, a rate of 40 determinations per hour could be attained. This is quite comparable with currently available automated systems. T o minimize sample preparation, a chemical derivative of cholesterol was not prepared. This is not common practice since derivatization increases the thermal stability and volatility of the molecule by decreasing its polarity. Consequently, it was necessary to determine and control those factors which promoted the thermal and/or catalytic decomposition of this steroid molecule. Sensible decomposition of cholesterol has been associated with both the injector port and the column. The carbon film formed in the injector port with use does not appear to promote decomposition. However, when an aliquot of the alkaline aqueous-alcoholic media was accidently introduced during sample injection, decomposition of the subsequent cholesterol samples was always noted. The chromatographic pattern associated with this situation exhibits decreased peak height ratios, cholesterol to internal standard, for all samples and standard with very pronounced tailing of the cholesterol peak. This condition was usually eliminated by cleaning the injector port with a piece of absorbent paper soaked with acetone or ether, but the introduction of this alkaline media may also ruin the column. On occasion when the cholesterol analyses were preceded by drug analysis, specifically urine extracts dissolved in solvents more polar than heptane, decomposition of cholesterol in the injector port has been noted. Cleaning the injector port has always eliminated this problem, and to date the columns have never been affected by chromatographing urine extracts. For decomposition associated with the column, the chro(17) L. P. Cawley, B. 0. Musser, S. Campbell, and W. Faucette, Amer. J. C/iti. Parlrol., 39, 450 (1963). (18) H. C . Curtius, W. Buergi, and K. Keller, 2. Kliit. Chem., 4, 38-42 (1966).
(19) J. A. Schmit and A. Mather, paper presented at the 16th Annual Meeting of the American Society of Clinical Chemists, Boston, Mass., Aug. 17-20, 1964. 1198
matographic pattern also exhibited diminished peak height ratios and, in addition, a third peak appeared as a shoulder on the internal standard peak. This source of decomposition occurred only when the column packing was heated during preparation or when a column was removed from the instrument when not at ambient temperature. T o date only in these cited instances has the decomposition of cholesterol been observed, and from our investigations there were no serious obstacles to daily operation. On the average the column life has been better than two months. Some insensible decomposition of cholesterol may occur, but if this is the case it must be a constant fraction or amount of the quantity injected since the calibration was linear. The fact that the peak attributed to cholesterol was cholesterol and not some decomposition product was substantiated by the following experiment. A glass capillary tube was placed in the flame tip of an unignited detector and then numerous injections of a heptane solution containing 600 mg cholesterol were made into the column connected to this detector. When the presence of a white residue was evident in the capillary tube, an IR spectra of this material was obtained, which was identical to that of cholesterol. Sensitivity. Under the present laboratory situation we have found that using the peak height ratio method (cholesterol to internal standard) was the most convenient measurement for calibrating the chromatographic data with serum concentration. As a result, in addition to the condition of the detectors, carrier gas, hydrogen, and compressed air flow rates, the resolving ability of the columns greatly affects the sensitivity. With the 2-foot columns sufficient resolution was available so that 1, 2, and 3 mpg of cholesterol gave easily detectable response at moderate electrometer settings as shown in Figure 2. This response and the peak height ratios were also affected by column absorption, which was controlled but not eliminated. Control was maintained by initially using multiple injections of a saturating solution containing 300 mg Z of cholesterol prior to standardization and then by maintaining the sample introduction at constant time intervals. Of the two compounds n-octacosane was minimally subject to column absorption. This is one reason for its selection as an internal standard and this proved of value in this investigation. The sensitivity, defined as the slope and intercept for the calibration, remained constant during the course of a series of analyses; however, when the time interval between sample injections was interupted, as occurred when samples were not continually added to the automatic sampler, saturation and standardization were repeated. During the course of a series, evaporation of the heptane from the extracts resulted in increased peak heights but not peak height ratios. The range of the slopes noted for the columns used to date was 4.5 f 0.5 X per mg % with negative intercepts that had numerical values corresponding to a serum concentration of 20 h 5 mg %, the limit of detection. The sensitivity from day to day was rather constant but after changing a septum or tank of carrier gas slight changes have been noted. In general, the initial sensitivity exhibited with a new column gradually decreases with time but never beyond the limits cited above. To date there has been no need to alter the hydrogen or compressed air flow rates to improve sensitivity. Accuracy and Precision. The insolubility of cholesterol and its esters does not permit making synthetic samples which were identical to serum. In this study pyridine was used as the solvent for cholesterol, cholesteryl palmitate, and
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
stearate. Ten replicate determinations were conducted o n solutions of each individual compound by adding 0.500 ml of sample and 0.500 ml of a 7 gram % albumin solution t o the alcoholic potassium hydroxide solution. The average per cent recoveries obtained on solutions containing the equivalent of 300 mg of cholesterol were: cholesterol 99, palmitate 98.5, and stearate 97.5. Initially 1-hour saponification, as in the Abell-Kendall method (16) was used, but often the recoveries for the esters were less than those cited above because of incomplete saponification. With the 3-hour reaction time, recoveries were always 97 % or better. With additional stirring, complete saponification was obtained with the shorter reaction time; however, we have chosen the 3-hour reaction time since it fits into our laboratory routine. Allowing the saponification to proceed as long as 48 hours a t room temperature does not affect the gas chromatographic analytical result. To determine the precision of the method, replicate determinations were conducted at three concentrations. The results were 110 + 7, 205 + 10, and 300 i 10 mg which correspond t o coefficients of variation of 7, 5 , and 3%. To date, we have detected values as low as 75 and as high as 800 mg %. For samples with values above the calibration range, 0.200 ml of serum was used in sample preparation. Operational Considerations. The average daily work load was 80 samples. Initially the injections were made manually, using two columns in the chromatograph, and it took approximately 2.0 hours t o complete the series. This was a very tedious task. With 1.0 hour for total sample preparations and 0.75 hour for calculations, it took approximately 4 man-hours to complete the series. The automatic injector eliminated the tedium and the method was then well accepted by the laboratory personnel. The daily man-hour requirement has not been reduced to approximately 2 hours. With the exception of cleaning injector ports and detectors, the gas chromatograph has not malfunctioned for over a year. During the same period, the electrode in the hydrogen generator had to be replaced once. The automatic sampler has been in use for over 10 months and we have had only one maintenance problem which prevented its use for 3 days. When purchased, the cable on the injector housing was too tightly clamped and with wear the wires eventually snapped. This problem has been eliminated by the manufacturer. The performance of the integrated instrument has made automated gas chromatography a reality in our laboratory. The initial expense for the equipment was competitive with other automated systems. With regard t o expendable items and reagents needed t o perform the 20,000 analyses completed to date, the G L C method was more economical than a single channel AutoAnalyzer. Comparative Data. We have compared the results obtained by the gas chromatographic method with the AbellKendall method. One hundred samples over a period of a month, of hospital patients and participants in the Multiphasic Screening Program were included in this set. Because we had noted poor recoveries of cholesterol from our synthetic samples with 1-hour saponification, the samples analyzed by the Abell-Kendall method were well-mixed at least twice during the saponification t o minimize the partial hydrolysis of esters. All analyses were run o n fresh serum and these results are listed in Table I. I n general, the Abell-Kendall and G L C methods agreed within plus or minus 10 mg %. However, we have observed differences for 5 samples of 40 t o 50 mg between the two determinations which were not explained by experimental error. The sera in question were
Table I. Comparative Cholesterol Data from the Analyses of 100 Random Samples Method Abell-Kendall GLC Mean mg 213 21 1 Standard deviation 58 51 Correlation coefficient for GLC with A-K ... 0.973 not lipemic, jaundiced, nor did they exhibit any unusual characteristics. Information concerning possible interferences from drugs was not available and at present n o explanation can be offered for these differences. We have also periodically compared the SMA 12/60 and G L C data over the last ten months and in more than 1000 duplicate determinations, the average G L C data was 1 2 x lower. G L C analyses were also conducted on 4 different lots of the SMA 12/60 Reference Serum, with listed assay values of from 247 to 252 mg % cholesterol. The G L C values obtained for these Reference Sera were between 210 and 220 mg %, an average of 14% lower than the listed assay. Since the slope for the calibration of the SMA 12/60 cholesterol channel was solely dependent on the listed cholesterol value for the Reference Serum, the difference between this value and the G L C value readily explained the average difference between the SMA 12/60 and GLC data. F o r approximately 50 samples the SMA 12/60 value was 50 to 75% higher than the G L C value which could not be explained by the common causes of spurious results with AutoAnalyzer systems such as carry over, base-line drift, and serum turbidity. Again, information concerning possible interferences due to drugs was not available and reasons for these inconsistent results are not available. We are, however, currently involved in a study under the direction of the Communicable Disease Center for the evaluation of cholesterol standards and hope that answers t o these discrepancies will be forthcoming. Normal Values. Over the last 16 months of operation the minimum age limit for the participants in the Multiphasic Screening Program was 40 years. This population represents a large fraction of the currently existing data and only the normal ranges covering this segment will be reported now. Normal ranges were determined, 95 confidence limit, by plotting the cumulative frequency per cent L'S. the observed values on probability paper. This treatment of the data, attributed t o Hoffman (20), was chosen for comparative purposes and because the sample populations closely approximated the normal distribution. I n Table I1 these results and the values reported by Henry ( I ) are listed. The values cited by Henry represent a summation of a number of studies conducted in Europe and the United States, including a joint study (21)in which the following laboratories participated : Cleveland Clinic Foundation; Donner Laboratory, University of California; Department of Nutrition, Harvard University; and Department of Biophysics, University of Pittsburgh. I n this joint study the method of choice for the cholesterol determination was the Abell-Kendall, since only with this method could consistent results be obtained among the four laboratories. One of the data representations published from this study was the mean cholesterol value for the 40-59 age group, the segment of society most subject t o coronary difficulties, and the (20) R. G. Hoffman, J. Amer. Med. Ass., 185, 864-873 (1963). (21) L. A. Lewis. F. Olmsted, I. H. Page, E. Y . Lawry, G. V. Mann, F. J. Starr. M. Hanig. M. A. Lauffer, T. Gordon, and F. E. Moore, Circulation, 16, 227 (1961).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
1199
a
Age 40-49 50-59 60-69 For GLC method.
Table 11. Age Dependent Normal Ranges Male Range mg % - Sample R a n GLC Henry population" 140-320 135-315 1775 145-330 145-340 1956 13C-310 14C-321 1228
Sample population" 1304 1418 761
g
Female e GLC 140-295 155-340 150-340
Table 111. Cholesterol Statistics on the 40-59 Age Group from Five Laboratories GLC Harvard" Donner Cleveland Pitts RIH 233.8 240.4 Mean mg % 242.4 241.9 241.2 48.2 46.4 Standard deviation 45.0 49.2 47.0 Sample population 683 2722 e 34 I450 1047 a Included was the Framingham Study with an average value of 239 mg %. Table IV. Frequency Distribution of Cholesterol Data for the 40-59 Age Group
Mg
z
100-119
120-1 39 140-159 160-179 180-199 2 W 219 220-239 240-259 260-279 280-299 300-319 320-339 34&359 360-379 380-399 400-4 19 420-439 440-459 Mean Std dev Total Coefficient of the moment of skewness
Male Summation of joint study 7 22 69 197 408 595 745 689 521 347 203 I06 47 28 14
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Female RIH
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29 95 206 329 454 439 412 292 204 136 55
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12 36 126 260 419 588 620 557 418 285 174 114 60 28 19 8 3 4 237.5 50.7 3731
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GLC RIH 237.5 50.7 3731
dall method should be considered more accurate. However, evaporation of the extracting solvent during preparation can produce small positive errors if not controlled, to which the GLC method was less susceptible because of the presence of an internal standard. Thus the G L C method was subject to a small negative error and the Abell-Kendall method, a small positive error, and together easily account for the difference in question. Results for Free Cholesterol. Few requests have been made for the determination of free cholesterol and our current data are limited. The chromatograms for free cholesterol determinations were identical t o those in Figure 2 and n o additional background has been noted. From other investigations, the elution of CI6or C18triglycerides or cholesterol esters from the column at the established conditions has not been observed. However, repetitive injections of samples containing these materials can slightly decrease the efficiency of the column. This does present a problem since by reconditioning the column at 265 " C for 1 hour the sensitivity is restored. CONCLUSIONS
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0.350
0.372
results from the four laboratories and our results are listed in Table 111. The frequency distributions for the G L C and the summation of the joint study data for this same age group are listed in Table IV. From all considerations of the data, the G L C method produces slightly lower values than the Abell-Kendall method with the best indication represented in Table I V by the ratio of means of the two male populations, 0.968. From a t test of the data this 3.2% difference cannot be attributed t o sampling so the methods d o yield statistically different values. The close relationship between the two methods is clearly demonstrated by the magnitude of the difference, and a consideration of the extraction steps in the two methods can explain the existence of a difference. In the AbellKendall procedure, 10.0 ml of extracting solvent are used instead of the 1.0 ml in the G L C method. With the larger volume of extracting solvent, the efficiency of the extraction should be greater for the former, substantiated by the reported recoveries and from which standpoint the Abell-Ken1200
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The method presented meets the criteria of an acceptable analytical procedure, since it has the property of specificity and primary standardization. In addition, it fulfills the requirements of a busy routine clinical laboratory, namely, reliability, ease of operation, high volume capability, and economy relative to equipment, reagents, and personnel. The accumulated data were consistent with results from comprehensive studies conducted over the course of years and demonstrate that automated gas chromatography is a reality. We have had experience with the automated Huang (22) method on a single channel AutoAnalyzer and the S M A 12/60 system. This method was particularly subject t o carry over and base-line drift and erratic results were common. The consequence of the lack of prime standards was reflected by the higher values obtained relative to the AbellKendall and G L C method. F o r these reasons the G L C method procedure is the method of choice in our laboratory. RECEIVED for review March 5,1971. Accepted April 22, 1971.
(22) T. C . Huang, C. P. Chem. V. Wefler, and A. Rafferty, ANAL. CHEM., 33, 1405 (1961).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
