Determination of Morphine and Codeine in Post-Mortem Specimens George R. Nakamura’ University of California, School of Criminology, Berkeley, Calif. 94 720
E. Leong Way University of California, School of Medicine, Deparfment of Pharmacology, San Francisco, Calif. 94 143
The finding of morphine in post-mortem specimens implicates heroin use. In suitable tissues or body fluids, its presence denotes acute intoxication and fatality. Wilkinson and Way ( 1 ) introduced a rapid, convenient procedure for the gas-liquid chromatographic (GLC) estimation of morphine in 0.1-1.0 ml of plasma or cerebrospinal fluid. A derivatization of the sample extract residue with trimethylsilyl ether (TMS) allowed sub-microgram estimation of morphine which would otherwise be difficult because underivatized bases tend to adsorb onto GLC columns in a nonlinear manner. Elliott et al. ( 2 ) also derivatized morphine with TMS in whole blood for quantitative purposes. These procedures were adapted and modified for determination of morphine and codeine in cadaverous body fluid and tissue samples. These extraction and derivatization techniques facilitated analyses of morphine and codeine in the free (unchanged) and bound (conjugated) forms in 4 grams of liver and ‘/z ml of bile or urine.
EXPERIMENTAL Reagents. Morphine sulfate USP, codeine sulfate USP, cocaine hydrochloride USP, and nalorphine USP were products of Mallinckrodt Chemical Works. Methadone hydrochloride USP was a gift from Eli Lilly Laboratories. The external standard mixture was prepared by mixing 0.1 mg per ml of each of these compounds, calculated as a free base, in methanol. The internal standard mixture was an aqueous solution of nalorphine HC1, USP, Merck, 0.1 mg per ml; each compound was calculated as the free base. A 40% solution of N,O-bis(trimethylsilyl)acetamide,or “BSA.” Pierce Chemical Company product No. 38837, was dissolved in dry pyridine (Pierce) and used as the silylating agent. The following grades of extracting solvents were used: methyl alcohol, GC-Spectrograde, Mallinckrodt; hexane, ethyl acetate, and chloroform, Nanograde, Mallinckrodt. The dipotassium phosphate powder was a product of J. T. Baker Chemical Co., ACS grade. Equipment. A Hewlett-Packard gas chromatograph Model 7610-A was equipped with two glass U-tube columns packed with 3% OV-17 on 100/120 Gas-Chrom Q and with 3.8% Silicon Rubber UC-W 98 on Chromosorb W.H.P. S O h O O mesh. The temperature of the injection port was 280 “C; the flame ionization detector was maintained at 280 “C, and the oven was held a t 240 “C. The carrier gas used was helium and it was maintained a t a flow of about 40 ml/min. (Nitrogen does not allow for a discrete separation of morphine and codeine in 3% OV-17.) All glassware was treated with 5% “Dri-Film Sc-87,” Pierce Chemical Company, in chloroform. Polypropylene ware was not treated. Procedure. Blood for Unbound Morphine. Into a 50-ml polgpropylene centrifuge tube, fitted with a screw cap lid, 4 ml of 40% K P H P O ~and 50 p1 of nalorphine internal standard were introduced. Then, 5 ml of whole blood was added and the tube shaken to mix the contents. T o this mixture, 25 ml of ethyl acetate:isobutanol, 9:1, was added and the tube was agitated on an Eberbach reciprocating shaking machine for 10 minutes. After the mixture was centrifugated a t 5000 rpm for 10 minutes, the supernatant was decanted into silicon-treated paper (WhatPresent address, County of Los Angeles, Department of Chief Medical Examiner-Coroner, 1104 No. Mission Rd., Los Angeles, Calif. 90033.
man 1PS) containing approximately 0.5 gram of sodium sulfate and collected in another 50-ml polypropyiene centrifuge tube. T o the filtrate was added 4 ml of 0.2N HC1 and the tube was shaken for 10 minutes. The top layer was aspirated and discarded and the bottom layer was washed twice with 10 ml of hexane which was aspirated after centrifugation. The remaining aqueous layer was evaporated rapidly in a Buchler Evapomix a t 30 “ C or allowed to evaporate overnight under a jet of air stream a t ambient temperature. The residue was dissolved in methanol and transferred quantitatively to a glass-stopped 3-ml centrifuge tube using methanol. The methanolic extract was evaporated to absolute dryness under a jet stream of air in a heating block (Roeco “Constantemp”) set a t 80 ‘C. The residue was then dissolved in 50 4 of BSA reagent using a Vortex-type mixer as an aid. After 3 minutes and within 30 minutes, 1 p1 was injected into GLC under standard conditions. The GLC system was calibrated before and after a series of determinations by injecting the external standard containing 0.05 pg each of methadone, morphine, codeine, cocaine, and nalorphine. This standard was made by evaporating 50 11 of the stock solution listed under “Reagents” in a heating block, the residue was taken up in 100 pl of BSA and 1 pl was injected under standard GLC conditions. This procedure allowed for a check t o determine if all components of the GLC were operating properly, Blood (tor Free plus Conjugated Morphine). Into a 100-ml capacity 25-mm X 200-mm round-bottom glass tube, equipped with a Teflon-lined screw cap, 5 ml of blood was introduced. Fifty p1 of nalorphine internal standard and 5 ml of 4090 HC1 was added and qixed in turn. The mixture was placed in an autoclave at 15-lb pressure for 15 minutes. After cooling, the pH of the mixture was adjusted to 9.0 with 40% NaOH. Approximately 0.5 gram of sodium carbonate:sodium bicarbonate 3:8 mixture was added. T h e extraction was conducted with 35 ml of ethyl acetate:isobutanol 9:l mixture on an Eberbach shaker for 10 minutes. The mixture was centrifuged for 15 minutes and the supernatant was filtered through Whatman 1PS paper containing approximately 0.5 gram of powdered sodium sulfate. The filtrate was collected in a 50-ml polypropylene tube, extracted into 0.2N HC1 which was washed with hexane in the same manner as for the analysis of unbound morphine. The GLC analysis conditions were the same as previously described. Bile and Urine. In a 15-ml centrifuge tube, 1 ml of bile or urine, 200 p1 of nalorphine internal standard, and 1 ml of 1.V H2S04 were mixed. The tube was loosely capped. The mixture was autoclaved for 15 minutes a t 15 pounds. Upon cooling, the hydrolysate was centrifuged for 5 minutes and 1 ml of supernatant was transferred to a 50-ml centrifuge tube. The pH of the aliquot was adjusted approximately to 9.0 with 40% NaOH, and the mixture then saturated with 8:3 mixture of NaHC03:Na*CO3. The p H was checked with p H indicator paper. Then, 25 ml of an ethyl acetate:isobutanol, 9:1, mixture was introduced and extraction was conducted on a mechanical shaker for 10 minutes. The remaining steps, after the shake-out,, were the same as for blood. Lioer and Kidney. Ten grams of tissue were homogenized in 20 ml of 30% HCI with a Brinkmann Polytron Homogenizer. Then 400 p1 of nalorphine internal standard were added to the mixture. The homogenate was autoclaved a t 15 lb for 15 minutes. Upon cooling, the total volume was measured. A 10-ml aliquot was transferred to a 100-ml capacity 25-mm X 200-mm round-hottom glass tube with a Teflon-lined screw cap. Forty per cent NaOH was used to adjust the p H to approximately 9.0 and approximately 0.5 gram of carbonate-bicarbonate powder mixture was dissolved in the A N A L Y T I C A L CHEMISTRY, VOL. 4 7 , NO. 4. A P R I L 1975
775
le COC
MO
cac
I
b 1
2
6
4
2
8 RETENTION TIME,
1
1
4
6
minutes
Figure 1. GLC separation of TMS derivatives of morphine, codeine, cocaine, and nalorphine on ( a ) 3.8% UCW-98 and ( b ) 3.5% OV-17 umns. Morphine and codeine reverse sequence of elution in these columns
COI-
M
Lr m z
P
h
Ln
ak N
CL w
a
.--I
--__A__-2
4
6
2
RETENTION T I M E ,
4
minutes
2
4
Figure 2. GLC profiles of morphine TMS (nn) in a, blood: b, liver: and c, bile. Nalorphine TMS ( N ) is used as an internal standard. OV-17 column used
mixture. The resulting mixture was extracted with ch1oroform:isobutanol, 8:2, on a mechanical shaker. The tube was then centrifuged for 10 minutes a t 3000 rpm. The top aqueous layer was aspirated off. The bottom layer was passed through a Whatman 1PS paper into a 125-1111 separatory funnel containing 5 ml of 0.2iL’sulfuric acid. The funnel was shaken on a mechanical shaker for 10 minutes. T h e lower solvent layer was drawn off and discarded. The aqueous layer was treated with IN NaOH to adjust the p H to approximately 9.0. A pinch of 0.5 gram carbonate-bicarbonate was added to the 776
ANALYTICAL CHEMISTRY, VOL. 47, N O . 4, APRIL 1975
mixture, which was then shaken with 25 ml of ethyl acetate:isobutanol 9:l. The lower aqueous layer was removed and discarded, and the upper layer was passed through Whatman 1PS paper containing approximately 0.5 gram of sodium sulfate into a 50-ml polypropylene tube containing 4 ml of 0.2N HCI. The tube was shaken for 10 minutes and the upper solvent layer was removed and discarded. The remaining steps, following the shake-out, were the same as for blood. In preparation for GLC injection, the final residue was dissolved in 100 p1 of BSA solution instead of 50.
Table I. Determination of Optimal Conditions for Acid Digestion of Blood a n d Liver Blood % HCIa
0 5
Table 11. List of BSA-Treated Narcotics and 0ther Toxicologically-Important Compounds 390 OV-17, 240 ’C
RRT‘
3.3:. UCW-98, 230 ‘C
Pentazocine Methadoneb Levorphanol Propoxypheneb Cocaineb Scopolamine Morphine Morphine glucuronide Codeine Normorphine Nor codeine Nalorphine Dihydromorphinone Diazepam Monoacetylmorphine Hydrocodoneb Oxymorphoneb Oxycodone Chlordiazepoxideb Diacetylmorphineb Papavarineb
0.26 0.26 0.27 0.29 0.42 0.52 0.69 0.73 0.74 0 .a2 0.87 1.oo 1.04 1.05 1.ll 1.13 1.31 1.42 1.57 2.03 2.33
Methadone Pr opoxyphene Cocaine Levorphanol Pentazocine Diazepamb Scopolamine Hydrocodone Norcodeine Codeine Morphine glucuronide Dihydromorphinone Chlordiazepoxide’ Oxycodone Morphine Normorphine Monoacetylmorphine Oxymorphone Diacetylmorphine’ Nalorphine Papavarineb
Liver,
Morphine mg %
Codeine mg %
Morphine mg %
0.03 5 0.064 0.076 0.090 0.085 0.073
0.122 0.120 0.143 0.150 0.148 0.138
...
0.62 10 0.64 20 0.66 25 0.71 30 0.66 40 ... ... 0.61 ... ... 0.42 50 a 70HC1 denotes final concentration of acid in mixture.
R E S U L T S A N D DISCUSSION Effect of Hydrolysis on Morphine Recovery. The comparative results obtained from the analysis of morphine and codeine in hydrolyzed and nonhydrolyzed blood samples are presented in Table I. The results indicate that as much as 2.5 times more morphine and 1.2 times more codeine can be determined from hydrolyzed samples. Table I also shows that the maximum yield was obtained for both morphine and codeine a t a final dilution of blood of 20% HCl. The disadvantage of digesting blood prior to analyzsis is that cocaine, methaqualone, diazepam, oxazepam, flurazepam, atropine, scopolamine, nisentil, pontocaine, methylphenidate, propoxyphene, methadone, and other drug compounds are labile in hot acid. There is a need for routinely examining chromatograms for forensically important drugs other than morphine or codeine; cocaine is a prime example. The obvious advantage of doing routine screening for unconjugated morphine in blood is in its simplicity and speed, especially when a large number of samples is involved. Assessment of Specificity f o r Morphine and Codeine Analyses. Figures l a and I b show the resolution of methadone, cocaine, codeine, morphine, and nalorphine in two different columns. The reversal of the order in which morphine and codeine are eluted in these columns was useful for identification purposes. The types of GLC profiles obtained from positive blood, bile and liver are shown in Figures 2a through 2c. Urine and bile extracts generally yield similar GLC profiles. More than 1500 post-mortem whole blood specimens were screened by the present GLC method for morphine and codeine by this procedure. Approximately 76% of these were determined. to be negative and eliminated from further testing. Some of these bloods were partially putrefied; however, the GLC profiles of the extracts presented no problem in their exclusion. Twenty-four narcotic compounds and drugs of toxicological interest and extractable as basic compounds were dissolved in BSA a t a concentration of 5 mg/100 ml and chromatographed in thch two standard GLC columns. Results are shown in Table 11. The present method affords a simultaneous analysis of forensically important compounds such as morphine, codeine, and cocaine from a specimen. Under standard analytical conditions, the minimum sensitivity for the detection of morphine was 0.005 mg per cent in blood, 0.03 mg per cent in liver and kidney, and 0.05 mg per cent in urine. BSTFA (N,O-bis(trimethylsily1)trifluoroacetamide)was used for derivatizing morphine and codeine extracted in blood ( 2 ) . BSTFA required heating the reaction mixture for 30 minutes following silylation for optimal GLC re-
’
RRT in reference to nalorphine
‘I
KK?
0.25 0.27 0.27 0.29 0.37 0.42 0.44 0.56 0.59 0.59 0.60 0.64 0.66 0.69 0.71
0.72 0.79 0.80 0.91 1.oo 1.57
Does not form T l l S deriva-
tive.
Table 111. Recovery of Morphine and Codeine Added to Blood, Urine, Bile, a n d Liver Percent recovel);‘
Specimen
Blood
hvlorphine a n d codeine concn, mg 5:
0.005 0.04 0.12 0.20
.Morphine
Codeine
55 .O 64.9 65.4 67.9 __ Av 63.3 (It4.93) 67.8 65.4 69.5 __ Av 67.6 (i2.83) 68.8
69.6 78.8 80.3 82.1 __ 74.1 (14.82) 83.6 Urine 0.10 0.30 84.7 0.50 85.6 __ 84.6 (io.82) Bile 0.10 85.5 0.30 70.5 87.0 0.50 72.5 86.3 69.2 1.oo 86.2 __ Av 70.3 86.2 (io.92) (10 .53) 56.9 Liver 0.05 75.4 62.2 0.10 79.8 64.3 80.9 0.20 61.5 0.50 80.2 __ Av 61.2 79.1 k2.77) b2.06) Mean value from triplicate determinations. Percent standard deviation in parentheses. ~
sponse. We observed that the same response for morphine and codeine can be obtained with BSA without heating. Maximal response was obtained in 3 minutes and was shown to be stable for 30 minutes. The spectrofluorometric procedure used for confirmation purpose was essentially that described by Kupferberg et al. ( 3 ) . This procedure was not used for routine drug search purposes because of its inherent lack of specificity. Pentazocine, nalorphine, and dihydromorphinone interA N A L Y T I C A L C H E M I S T R Y . V O L . 47, NO 4 , A P R I L 1 9 7 5
* 777
fered with morphine readings, as previously noted by Passwater ( 4 ) . Codeine does not interfere under conditions used for the analyses. However, if body fluids or tissues from a codeine fatality were tested, one would obtain readings for morphine, which is one of the biotransformation products of codeine. Thirty random blood samples which were found positive for morphine by spectrofluorometric analysis were selected and the results were compared by GLC determinations. A qualitative correlation was shown in every case because each positive GLC value for morphine was confirmed by a positive fluorescence. A correlation coefficient of 0.94 which was calculated for the two sets of data appeared to give a favorable overall agreement. Calibration. Standard curves for morphine and codeine in blood were linear over the concentration range of 0.02 t o 0.12 Mg. Linear plots were also obtained from the extraction of these compounds in liver, bile, and urine in the concentration range of 0.5 to 2.5 Mg. Table I11 shows the range of recovery of morphine and codeine from the various specimens.
Since morphine, codeine, and the internal standard (nalorphine) were added to these specimens to obtain the data, these curves afforded analytical values which included a recovery factor for each of the tissues run. T o offset any changes in peak area over a time span, recovery tests on blood and other tissue samples containing known amounts of added morphine and codeine were periodically run to obtain a nalorphine/morphine value to be used as a calculation factor. LITERATURE CITED (1) G. R. Wilkinson and E. L. Way, Biocbem. Pbarmacol, 18, 1435 (1969). (2) H. W. Elliott, K. D. Parker, J. A. Wright, and N. Nomof, Clin. Pbarmacoi. Tber., 12, 806 (1971). (3) H. Kupferberg, A . Burkhaiter, and E. L. Way, J. Pbarm. Exp. Tber., 145, 247 (1964). (4) R. A. Passwater. "Fluorescence News," American instrument Go., Silver Springs, Md., 6, 8 (1971).
RECEIVEDfor review September 9,1974. Accepted December 11,1974.
I CORRESPONDENCE Determination of Low Surface Areas by the Continuous Flow Method Using an Interrupted Flow Technique Sir: In a previous publication ( I ) , we described our improved continuous flow method. A shoulder was sometimes obtained high on the leading edge of the desorption peak. Unfortunately, we did not rigorously define the conditions under which we obtained these shoulders and this misled Lowell and Karp ( 2 ) into the assumption that they were characteristic of peaks obtained from samples of low surface area. This was not so and, in the present communication, it is hoped to resolve this and also to describe a method for the determination of low surface areas. EXPERIMENTAL The apparatus was similar to that described in reference ( I ) with a few exceptions. For rapid turnover of control samples, individual metering of the gases was replaced by premixed cylinders of helium and nitrogen. Two-way valves (Drallim Couplings Ltd. Whyteleafe, Surrey) were provided each side of the sample tube so that it could be isolated from the gas stream. The practice of cooling a tube in the reference stream a t the same time as the sample was discontinued. The single point method at a relative pressure of 0.2 was used. The sample tube had side arms of 2-mm internal diameter and a region 3.5 cm long of 13-mm internal diameter to contain the sample. The flow rate used was 15 ml/min. The adsorbent used was crystallized unmilled ammonium perchlorate obtained from three different sources and one milled sample of higher surface area.
RESULTS AND DISCUSSION Problems have arisen previously in the measurement of low surface areas (2-4). Similar problems occurred in the present case, as shown by Figure l a . However, the shoulder referred to in ( I ) was obtained only on an alumina sample. This had a surface area of 67 m2/g and the measurements were performed on a sample weighing 0.0960 g. The volume of gas adsorbed ranged from 1.24 to 1.97 ml, depending on the relative pressure, and the measuring system was attenuated by a factor of more than thirty below the level a t which anomalous peaks became clearly visible. 778
ANALYTICAL CHEMISTRY, VOL. 47, N O . 4, APRIL 1975
With the type of sample tube used in the present work, which allowed the use of a reasonable sample size, anomalous peaks were of no importance in measurements on samples having surface areas greater than about 0.2 m2/g. Unmilled ammonium perchlorate has a very low surface area (
