ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
2145
Reproducibility of the Internal Standard Method in Gas Chromatographic Quantitation of Cocaine Sir In gas chromatographic (GC) quantitation, the method of preference involves use of an internal standard. T h e internal standard method should compensate for variations and inaccuracies in t h e volumes of aliquots delivered into the chromatograph. Recently there has been some recognition of the fact that the internal standard method may not be so reliable ( I , 2). However, very little has been suggested as to the basic causes for the observed variations in results obtained using t h a t method. T h e work of Shatkay and Flavian ( I ) shows that the ratio of analyte to internal standard ( R J varies considerably with change in volume injected for t h e system trimethyl phosphate/dodecane in methanol. Less variation was encountered in a n undecane/dodecane system. In contrast to that work, the letter of Royston (2) noted no significant variation of R.4 with volume injected for the system n-nonaneln-decane in CHCI,. T h e reason for this contradiction may he perceived from t h e results presented in the following study. This study explores a situation arising in the use of the internal standard method in GC quantitation of cocaine. The procedure in question involved a standard solution of 1 m g / m L of cocaine HCl in CHCI, with 0.5 m g / m L of either methyl palmitate or eicosane as internal standard. Aliquots of the standard solution were limited to 0.5 pL size t o accommodate the sensitivity of the flame ionization detector to large amounts of solvent and to increase column life. On occasion, serial identical aliquots of standard solution injected would give peak area ratios of cocaine t o internal standard which varied as much as 50%. At other times. no such problem was encountered. An investigation was carried out to discover the cause of t h a t behavior and a remedy for t h e problem. T h e behavior of the alkaloid salt was investigated, since the hydrochloride must be thermally decomposed on t h e GC column in order for t h e cocaine to elute as the free base. This thermal decomposition might be a source of variation in quantitative results. In addition, some observations were made on the possible interactions of both solutes and the solvent in t h e standard solution.
EXPERIMENTAL Gas chromatography *as carried out on Hehlett-Packard 5830 and 5840 instruments. These chromatographs are microprocessor controlled and print out cocaine and internal standard peak areas. The ratios of those peak areas were then determined using a Texas Instruments SR-51A calculator. Means, standard deviations with N 1weighting, and coefficients of variation (CV) were calculated on the peak area ratios by the follouing formulas: ~
cv =
SD ( 1 0 0 % )
x
(3)
where X is the peak area ratio and ?I is the number of ratios averaged. Internal standards, eicosane, tetracosane, and methyl palmitate, were supplied by Applied Science Laboratories, Inc. Cocaine HC1 and procaine HC1 were obtained from Merck & Co., Inc. Trihexiphenidyl HC1 was obtained from Lederle Laboratories. 0003-2700/78/0350-2145$01 O O / O
Table I. Effects of Free Base. Different Internal Standard,-and Concentration X (N = conditions SD 10) 1 . 0 4 0.105 1. 220 "C, OV1, 5-pL syringe, 0.2 p L of cocaine HC1:eicosane 1:0.5 0.040 1.03 2. 220 "C, OV1, 5-pL syringe, 0.2 p L of cocaine base:eicosane 0.89:0.5 3. 220 'C, OV1, 10-pL syringe, 1 . 0 8 0.014 1.0 p L of cocaine HC1:eicosane 1:0.5 4. 220 'C, OV1, 10-pL syringe, 1 . 0 8 0.006 1.0 p L of cocaine base:eicosane 0.89:0.5 1.04 5. 220 "C, OV1, 5-pL syringe, 0.069 0.2 p L of cocaine HC1:eicosane 1 : 0 . 5 with diethylamine added 6. 220 '(2, OV1, 10-pL syringe, 1.09 0.040 1.0 p L of cocaine HC1:eicosane 1 : 0 . 5 with diethylamine added 1.03 0.106 7. 250 'C, OV17, 5-pL syringe, 0.2 p L of cocaine HC1:tetracosane 1:0.5 1.03 0.041 8. 250 'C, OV17, 10-pL syringe, 1 pL of cocaine HC1:tetracosane 1:0.5 1.01 0.089 9. 270 "C, OV17, 5-pL syringe, 0.2 pL of cocaine HC1:tetracosane 1:0.5 0.020 10. 270 'C, OV17, 10-pL syringe. 0.99 1 . 0 p L of cocaine HC1:tetracosane 1: 0.5 0.20 0.004 11. 220 "C, OV1, 10-pL syringe, 1 p L of cocaine HC1:eicosane 0.2:O. 5
CV, %
10.1 3.9 1.3
0.6 6.7
3.7
10.3
4.0
8.8 2.0 2.1
Injections on the gas chromatographs were made with 5-pL and 1O-iL Hamilton Co. syringes. The injections were made directly onto l, .I inch X 1 ft OV1 or OV17 columns supplied by Hewlett-Packard.
RESULTS AND DISCUSSION Small aliquots (0.2 pL) were injected in some of the experimentation because the effects of parameter changes were more pronounced a t those volumes. Line 1 in Table I illustrates the large variation experienced under the most unfavorable conditions. Lines 2 through 6 were the result of an attempt to see if the problem was caused by inefficient conversion of cocaine salt to free base on injection. A cocaine solution was used for lines 2 and 4 in which there was no salt of the alkaloid. .4marked improvement in standard deviation was attained. However, when diethylamine was added to a standard solution of cocaine HCI to achieve the same end, no real improvement was noted. Further addition of diethylamine increased the standard deviation. Higher column temperatures could be expected to aid in problems of inefficient vaporization. Lines 7 through 10 show the effect of different column packing and internal standard to allow higher operating temperatures. Improvement was noted a t 270 OC on t h e O W 7 column, but not sufficient to consider the problem solved. U p to this point, the injection procedure consisted of drawing a n aliquot of solution into the syringe, inserting the C 1978 American Chemical Society
2146
ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
Table 11. Effects of Less Analyte and Internal Standard in Needle -
conditions 1. 220 'C, O W , 5-pL syringe, 0.2 p L of cocaine HC1:eicosane 1:0.5, air in needle 2. 220 'C, OV1, 5-pL syringe, 0.2 p L of cocaine HCI:eicosane 1:0.5, only tip of needle in septum 3. 220 'C, OV1, 5 p L syringe, 0.2 p L of cocaine HC1:eicosane 1:0.5 and 1 p L CHCl, 4. 220 OC, OV1, 5-pL syringe, 0.2 p L of cocaine HC1:eicosane 1:0.5 and 1 pL CHCl,, only tip of needle in septum 5. 220 "C, OV1, 1 O - p L syringe, 1.0 p L of cocaine HC1:eicosane 1 : 0 . 5 and 1 U L CHC1,
X(N=
cv,
1.07
SD % 0.052 4.9
1.00
0.032
10)
3.2
1.10
0.036
3.3
1.05
0.032
3.0
1.13
0.017
1.5
needle fully onto t h e column, and depressing t h e syringe plunger. A better procedure was used in which drawing u p a n aliquot of standard solution was followed by further drawing up 0.5 pL of air prior to injection. Improved results were noted (Table 11, line 1). With those results in mind, a series of injections were made with the former procedure except that t h e needle was allowed to barely penetrate the injector septum of the chromatograph before rapid injection of t h e solution. Even more marked improvement resulted. In a similar experiment, a series of injections were made with 1 pL of CHC13 pulled into the syringe after the standard solution so as to have no cocaine HC1 in the needle at the time of injection. T h e results of this effort are shown in lines 3 through 5 of Table 11. T h e expected decreases in standard deviation were achieved. Apparently much of the problem stems from the difficulty of washing all of the cocaine HC1 out of the syringe needle on injection. T h e last series of injections were of a mixture of 1.0 mg of cocaine HC1. 1.45 mg of procaine HCl, 1.06 mg of trihexiphenidyl HC1, and 0.5 mg of methyl palmitate per 1m L of CHC1,. Procaine HC1 is less soluble, methyl palmitate is more soluble, and trihexiphenidyl HC1 is about equally soluble in CHC13 with respect to cocaine HCl. T h e results of t h a t experiment are displayed in Table 111. Injections in the series in Table I11 were made by drawing u p 0.3 pL of the solution into the syringe, inserting the needle fully into the chromatograph, and immediately depressing the plunger. No improvement was seen with internal standard of solubility equal to cocaine HCl, b u t the large SD with procaine HC1 internal standard convincingly illustrates the importance of solubility. I t is interesting to note that X for cocaine/procaine increases with decreasing aliquot size from line 4 t o line 1 of Table 111, indicating that less procaine is injected relative to cocaine. T h e opposite behavior is experienced with respect to methyl palmitate from line 5 to line 2 of Table 111. Probably an error producing solubility effect will not only decrease the reproducibility, but it will also change the X with a change in the volume injected. CONCLUSIONS T h e results given here do not show any significant interaction of internal standard and cocaine HCl in the vapor phase. Nor is any great difficulty indicated in thermally converting cocaine HC1 to cocaine-free base in a reproducible manner. T h e problem seems to stem mainly from the solubility of cocaine HC1 in CHC1, and occurs in the needle a t the time of injection. The mechanism appears to be that, on insertion of the needle into the hot injection port, the CHC1, in the needle is immediately boiled away. This leaves a deposit
Table 111. Effects of Differing Solubilities of Internal Standards F(N= conditions 11) SD 1. 0 . 3 p L injected, cocaine/procaine ratios averaged 2. 0 . 3 g L injected, cocaine/methyl palmitate ratios averaged 3. 0.3 p L injected, cocaine/trihexiphenidyl ratios averaged 4. 0.5 p L injected, cocaine/procaine ratios averaged 5. 0.5 p L injected, cocaine/methyl palmitate ratios averaged 6. 0.5 p L injected, cocaine/trihexiphenidyl ratios averaged
1 . 1 3 0.244
21.6
0.95
0.060
6.3
0.68
0.043
6.3
1.01
0.138 13.7
0.98 0.047
4.8
0.65
7.4
0.048
of cocaine HCl and internal standard on the inside of the needle which is then partially washed away on expulsion of the remainder of the solution by depression of the plunger. T h e washing process is not reproducible and gives rise to variance of results. Magnitude of variance in these quantitations then depends on the ratio of the volume inside the syringe needle to the actual volume of solution injected. Sometimes a syringe will deliver less actual volume of solution than is indicated by the distance traveled by the plunger on injection. This may be due to solid particles in solution blocking t h e needle, a bent needle, or a worn plunger. Standard solutions of t h e free base would give better reproducibility over a short period of time, but the base appears to be less stable than the hydrochloride salt. Lowering the amount of cocaine HC1 in t h e solution while keeping the internal standard concentration constant improves reproducibility. In addition, larger aliquot sizes are to be preferred with steps taken to ensure that no cocaine HCl remains in the needle after injection. These principles would appear t o be important in any quantitative gas chromatographic procedure involving an internal standard injected through a needle into a heated injection port. T h e results published in references ( I ) and ( 2 ) may be explained by these data. In ( I ) t h e phosphate ester should be more soluble in the polar solvent, methanol, than the internal standard, dodecane. Therefore, more of the analyte will be washed out of the needle during injection, resulting in higher values of R A as the volume injected decreases. In contrast, the nonpolar pair of undecane analyte and dodecane internal standard will exhibit similar solubilities in methanol with the longer chain alkane being slightly less soluble than the analyte, undecane. T h e result would be a variation of R A with volume injected of less magnitude but similar direction as in the ester/alkane pair. Different results were achieved in ( 2 ) because a different solvent, chloroform, was used. Both analyte and internal standard were so soluble in that solvent that no variation in R A was observed. Obviously the ideal situation is t h a t both analyte and internal standard are very readily washed from the syringe needle by the solvent. If that is not possible, the methods given in this paper will improve reproducibility. LITERATURE CITED (1) A . Shatkay and S. Flavian, Anal. Chem., 49, 2222 (1977). (2) G. C. Royston, Anal. Chem., 5 0 , 1005 (1978).
J. C. Roberson Georgia State Crime Laboratory, 959 East Confederate Avenue, Atlanta, Georgia 30316
RECEIVED for review July 5 , 1978. Accepted August 28, 1978.
