Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 21)

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Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 21)

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Introduction: There are a number of compounds, such as morphine and codeine, which are classified into the opium alkaloids (opiates). They are being used as ethical drugs of narcotic analgesics and antitussives; 1 % powder of codeine or dihydrocodeine is commonly included in over-the-counter drugs of antitussives. Figure 3.1 shows metabolic pathways of morphine, heroin and codeine. Since morphine and codeine are finally excreted into urine in the conjugated forms with glucuronic acid [1–3], it is necessary to hydrolyze the conjugated forms of these compounds before GC/MS analysis. Heroin is rapidly deacetylated and finally excreted into urine as morphine...

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Nội dung Text: Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 21)

  1. 2.3 II.2.3 Morphine and its analogues by Hideyuki Yamada and Kazuta Oguri Introduction There are a number of compounds, such as morphine and codeine, which are classified into the opium alkaloids (opiates). They are being used as ethical drugs of narcotic analgesics and anti- tussives; 1 % powder of codeine or dihydrocodeine is commonly included in over-the-counter drugs of antitussives. > Figure 3.1 shows metabolic pathways of morphine, heroin and codeine. Since morphine and codeine are finally excreted into urine in the conjugated forms with glucuronic acid [1–3], it is necessary to hydrolyze the conjugated forms of these compounds before GC/MS analysis. Heroin is rapidly deacetylated and finally excreted into urine as morphine glucuronides. There- fore, it is not easy to discriminate the heroin use from morphine use [4, 5]. The detection of 6-acetylmorphine is recommendable for diagnosis of heroin use, because of its relatively long half-life in the body [4]. For accurate diagnosis of a cause of death in an opiate poisoning case, the ratio of a free form to a conjugated form becomes important (see section 4 of this chapter). In such a case, an opiate before (free form) and after (a total amount) hydrolysis should be analyzed. The amount of a conjugated form can be calculated by subtracting the amount of a free form from the total amount. By HPLC, the simultaneous analysis of free and conjugated forms is possible without any hydrolysis; in the near future, LC/MS may become a main tool for analysis of opiates and their metabolites. However, at the present time, GC/MS is being widely used for opiate analysis. For HPLC analysis of the conjugated forms of opiates, the authentic standards of mor- phine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) are necessary. In U.S.A. and Europe, it is easy to obtain these authentic compounds from commercial sources, but the import of these compounds to Japan is strictly controlled; easing of import of such compounds should be realized. GC and GC/MS analysis Reagents and their preparation • Ethylmorphine (internal standard, IS) a solution: a 1-mg aliquot of ethylmorphine hydro- chloride (Sankyo Co., Ltd., Tokyo, Japan) is dissolved in purified water to prepare 1 mg/mL solution. A 0.1-mL aliquot of this solution is mixed with 1.9 mL of purified water for 20-fold dilution (the final concentration in the form of the hydrochloride salt: 50 µg/mL), which is stored at –20 °C. © Springer-Verlag Berlin Heidelberg 2005
  2. 196 ⊡ Figure 3.1 Morphine and its analogues Main metabolic pathways of morphine, heroin and codeine. The thick and thin arrows show the main and minor metabolic pathways, respectively.
  3. GC and GC/MS analysis 197 • Standard solutions of morphine for a calibration curve (2, 6 and 20 µg/mL): a 1-mg aliquot of morphine hydrochloride (Shionogi & Co, Ltd., Osaka, Japan and other manufacturers) is dissolved in purified water to prepare 1 mg/mL solution; 0.1 mL of this solution is mixed with 4.9 mL purified water for 50-fold dilution (20 µg/mL). The latter solution is diluted 3.33-fold and 10-fold to prepare 6 and 2 µg/mL solutions, respectively, which are also stored at –20 °C. For preparing the standard solutions to be used for a calibration curve of 6-acetylmorphine hydrochloride or codeine phosphate (Shionogi & Co.), the same dilution procedure is followed. 6-Acetylmorphine hydrochloride can be synthesized by the method previously reported [6]. • 5 M NaOH solution (100 mL): a 20-g aliquot of NaOH is dissolved in about 70 mL purified water in a 100-mL volume glass beaker with stirring in an ice bath. After the temperature of the solution is cooled to room temperature, it is transferred to a 100 mL-volume volu- metric flask together with the water solution which has been used for washing the above glass beaker, and the volume is adjusted to 100 mL exactly. The solution is kept at room temperature; it is essential to seal the flask airtightly, because CO2 in atmospheric air can be absorbed into the NaOH solution to yield NaHCO3, resulting in the decrease of titer of the solution. • 5 M NH4Cl/NH3 buffer solution (pH 9, about 200 mL): a 23.7-mL volume of 28 % ammo- nia water solution is dissolved in purified water to prepare 250 mL solution (5 M NH3 solu- tion). A 13.4-g aliquot of NH4Cl is dissolved in purified water to prepare 50 mL solution (5 M NH4Cl solution). An appropriate amount of 5 M NH3 solution is mixed with the 5 M NH4Cl solution to adjust the pH to 9.0. The buffer can be stored at room temperature. • 10 M KOH solution saturated with KHCO3 (100 mL): a 56-g aliquot of KOH is dissolved in about 70 mL purified water in a 100-mL volume glass beaker with stirring in an ice bath. The volume of the solution is adjusted to about 85 mL with purified water and left until being cooled to room temperature. The KHCO3 powder is added to the NaOH solution until its saturation with stirring (about 30 g necessary). Upon saturation, the volume of the solution becomes to be about 100 mL. This can be used for experiments and is preservable at room temperature. • Pretreatment of a solid-phase extraction cartridge (Bond Elut Certify) b: a 2-mL volume of methanol and 2 mL purified water are passed through a Bond Elut Certify cartridge (Var- ian, Harbor City, CA, USA) just before use. For a new cartridge, each solution can flow by natural pressure through it without any aspiration. When the flow stops, it is aspirated for a moment; then the methanol or water can flow through it only by gravity. The purified water should not be passed through the cartridge completely; the cartridge should not be dried. The water flow should be stopped, when the water volume in the reservoir becomes small. Analytical conditions GC column: DB-1 (15 m × 0.25 mm i. d., film thickness 0.25 µm, J & W Scientific, Folsom, CA, USA). GC/MS conditions; instrument: an HP5890 GC-HP 5989A MS Engine (Agilent Technolo- gies, Palo Alto, CA, USA); column (oven) temperature: 200 °C (0.5 min) →5 °C/min →260 °C (3 min); injection temperature: 200 °C; injection volume: 1 µL (splitless mode); carrier gas: He; flow pressure: 6 psi; ionization mode: EI; electron energy: 70 eV.
  4. 198 Morphine and its analogues Procedures Two extraction methods, the liquid-liquid extraction and the solid-phase extraction, are avail- able; the latter gives cleaner extracts for GC and GC/MS analysis. i. Liquid-liquid extraction i. A 2-mL volume of urine c and 1.5 mL of concentrated hydrochloric acid are placed in a 50-mL volume glass centrifuge tube with a ground-in stopper, and heated at 100 °C for 30 min in a water bath to hydrolyze the conjugated forms of a target compound. The water level of the water bath should be slightly above the surface level of the hydrolysis solution in the tube. As a blank test, 2 mL urine obtained from a healthy subject is also treated as above. For quantitative experiments, in addition, a 0.1-mL volume each of solutions at three concentrations of an opiate (2.6 and 20 µg/mL) is added to 2 mL each of the blank urine; these samples are also processed in the same way as above. ii. After cooling to room temperature, 0.1 mL of the IS solution is added to the hydrolyzed solution. A 3-mL volume of 5 M NaOH is added to the solution for neutralization, followed by the addition of 4 mL of 5 M NH4Cl/NH3 buffer solution (pH 9). The final pH of the solu- tion should be checked with a test paper (Whatman, type CF); if the pH of the solution shifts from 9, it should be readjusted to pH 9.0 by adding the above ammonium buffer solution. iii. The above solution is extracted with 15 mL of chloroform/ isopropanol (9:1, v/v) by shaking. iv. After centrifugation at 3,000 rpm for 5 min, the organic (lower) layer is carefully trans- ferred to another 50-mL volume glass centrifuge tube of the same type with a pipette, fol- lowed by the addition of a sufficient amount of anhydrous sodium sulfate (2–3 g), and mixed well. v. The organic solution is passed through folded filter paper to remove the dehydrator and collected in a 10-mL volume glass centrifuge tube d with a ground-in stopper ( the shape of the tube bottom preferably to be conical). The solution is evaporated to dryness under a stream of nitrogen with warming the tube at 30–40 °C. vi. The residue in the tube is mixed with 50 µL of N,O-bis(trimethylsilyl)acetamide (BSA) rea- gente, capped airtightly and heated at 80 °C for 20 min; a 1-µL aliquot of the derivatized solution is injected into GC or GC/MS. vii. For the unconjugated (free) forms of morphine, codeine and 6-acetylmorphine, the extraction is made as follows f. A 2-mL volume of urine is diluted 2-fold with purified water and mixed with 0.1 mL of the IS solution and 0.2 mL of 5 M NH4Cl/NH3 buffer solution (pH 9). The pH of the solution is confirmed to be 9 with a pH test paper; if not, an appropriate amount of the ammonium buffer solution is added to it. The solution is extracted with 10 mL of chloroform/ isopropanol (9:1, v/v), and the following procedure is performed according to the above iv–vi steps. The blank urine and calibration samples are also processed in the same way. ii. Solid-phase extraction i. The hydrolysis of urine is made according to the step i of the above section. ii. The hydrolyzed solution is mixed with IS solution a, followed by a careful and gradual addi- tion of 1.5 mL of 10 M KOH solution saturated with KHCO3 (bubbling appears), mixed with 3 mL of 2 M Tris-HCl buffer solution (pH 8.1) and stirred well. The pH of the final solution is comfirmed to be 8–9 with a test paper. If not, the pH of the solution is adjusted to 8–9 using the 10 M KOH solution saturated with KHCO3 or 1 M HCl solution.
  5. GC and GC/MS analysis 199 iii. The solution is poured into a Bond Elut Certify Cartridge to let the solution flow inside the cartridge slowly taking more than 2 min to adsorb an opiate. iv. The cartridge is washed with 2 mL each of purified water, 0.1 M acetate buffer solution (pH 4) and methanol, and dried for 2 min by aspiration with a vacuum manifold, followed by washing with 3 mL methanol and drying for 5 min again. v. The target opiate is eluted, by passing 2 mL of dichloromethane/isopropanol/ammonia wa- ter (80:20:2, v/v) through the cartridge, into a 10-mL volume glass centrifuge tube with a ground-in stopper (the shape of the tube bottom preferably to be conical). The eluate is evaporated to dryness under a stream of nitrogen with warming at 30–40 °C. vi. The derivatization and injection into GC or GC/MS are made exactly in the same way as that described in the step vi) of the above liquid-liquid extraction section. vii. For the unconjugated (free) forms of morphine, codeine and 6-acetylmorphine, the extrac- tion is made as follows. A 2-mL volume of urine is diluted 2-fold with purified water and mixed with 0.1 mL of the IS solution and 0.2 mL of 2 M Tris-HCl buffer solution (pH 8.1). The pH of the solution is confirmed to be 8–9; if not, an appropriate amount of the above buffer solution is added to it. The blank urine and calibration samples are processed in the same way. These samples are equally treated according to the above steps iii–vi. Assessment and some comments on the methods Qualitative analysis is performed by finding a peak appearing at the same retention time as that of the authentic standard after trimethylsilyl (TMS) derivatization; it is also impor- tant to confirm the absence of the corresponding peak in the blank specimen. The final iden- tification is made by comparing a mass spectrum obtained from a test specimen with that obtained from the authentic standard. A total ion chromatogram (TIC) of the authentic com- pounds is shown in > Fig. 3.2; the mass spectra of the derivatized compounds are shown in > Fig. 3.3. Quantitation is performed by selected ion monitoring (SIM) using the peak height or area ratio of a test compound to IS; the ratio is applied to a calibration curve, which has been prepared in advance, to calculate the concentration of the test compound in a speci- ⊡ Figure 3.2 TIC of TMS derivatives of morphine and its analogues by GC/MS. Heroin is not derivatized. The peak of the TMS derivative of ethylmorphine is not included in this chromatogram; but it is eluted at 9.4 min under the same conditions.
  6. 200 Morphine and its analogues ⊡ Figure 3.3
  7. HPLC analysis 201 men. The peaks to be detected are as follows (the mass numbers underlined to be used for quantitation). morphine-TMS: m/z 429 (M+), 414, 401, 287 6-acetylmorphine-TMS: m/z 399 (M+), 340, 287, 204 codeine-TMS: m/z 371 (M+), 343, 234, 178 ethylmorphine-TMS: m/z 385 (M+) nalorphine-TMS: m/z 455 (M+) By SIM of GC/MS, a low level of morphine less than 100 ng/mL can be detected and quanti- tated; however, when a low level of morphine is detected, special care should be taken, because of the following reasons. Low levels (0.1–25 pmol/g tissue) of morphine exist in various ani- mals including humans as an endogenous compound; the levels are significantly increased by various stresses [7]. Therefore, the detection of low levels of morphine starting from a large amount of a specimen cannot be a proof of the intake of the drug. The level of morphine in urine of healthy subjects determined by GC/MS was reported to be about 1 ng/mL [8]. The poppy seeds are being used for various foods; when the foods including the seeds are eaten, an appreciable amounts of morphine (including its conjugates) are reported to be excreted into urine [9]. Because of these reasons, it seems reasonable to set a cutoff value of morphine in urine to be 300 ng/mL. However, caution is needed, because a morphine concentration higher than the cutoff level can be detected in some urine samples of subjects who have eaten a food containing poppy seeds [9]. It should be noted that an amount of codeine comparable to that of morphine are also included in the poppy seed- containing foods [10]. The half-life of heroin disappearance in human body was reported to be only about 2 min. It is, therefore, impossible to detect heroin itself from urine to prove its abuse. 6-Acetylmorphine is alternatively being analyzed as an indicator of heroin abuse; however, even with this metabolite, its half-life is only about 40 min, which is much shorter than that of unconjugated morphine (3.7 h) [4]. Even if the cutoff value of 6-acetylmorphine is set to be as low as 0.8 ng/mL, it is being consid- ered necessary to analyze a urine specimen sampled not later than 5 h after the heroin intake [4]. HPLC analysis Reagents and their preparation • Standard solutions of morphine for a calibration curve (2, 6 and 20 µg/mL): see “Reagents and their preparation”, above of GC and GC/MS analysis section. • 0.5 M Ammonium sulfate solution (pH 9.4, 100 mL): a 6.6-g aliquot of ammonium sulfate is dissolved in about 80 mL purified water, followed by the adjustment of its pH to 9.4 with dilute ammonia solution. The volume is adjusted to 100 mL using a volumetric flask and stored at room temperature. Mass spectra of TMS derivatives of the authentic morphine and its analogues. A: morphine-TMS; B: 6-acetylmorphine-TMS; C: codeine-TMS; D: ethylmorphine-TMS; E: nalorphine-TMS. A 50-µL volume of BSA is added to 20 µg each of opiates for derivatization [see the section 2-3)-(1)-vi)]; a 0.4-µL aliquot each is injected into GC/MS.
  8. 202 Morphine and its analogues • 5 mM Ammonium sulfate solution (pH 9.4): a 66-mg aliquot of ammonium sulfate is sub- jected to the above procedure, and stored at room temperature. • 0.5 M Phosphate buffer solution (pH 2.1, 500 mL): a 24.5-g aliquot of phosphoric acid is dissolved in about 400 mL purified water, followed by adjustment of its pH to 2.1 using several percent NaOH solution. The final volume of the solution is adjusted to 500 mL by adding purified water, and stored at room temperature. • 50 mM Phosphate buffer solution (pH 2.1, 100 mL): a 0.5-g aliquot of phosphoric acid is dissolved in about 80 mL of purified water, followed by adjustment of its pH to 2.1 using dilute NaOH solution. The final volume of the solution is adjusted to 100 mL by adding purified water, and stored at room temperature. • 0.2 M Sodium dodecyl sulfate (SDS) solution (100 mL): a 5.8-g aliquot of SDS is dissolved in about 70 mL purified water in a volumetric flask, and left overnight to dissipate bubbles. Purified water is gently added to the above solution to prepare 100 mL solution, mixed well and stored at room temperature. • Mobile phase, 5 mM-SDS containing 100 mM phosphate buffer solution (pH 2.1)/ace- tonitrile (76:24, v/v, 1 L): a 152-mL volume of 0.5 M phosphate buffer solution and 19 mL of the above 0.2 M SDS solution are placed in a 1-L volume glass graduated cylinder; the final volume of the solution is adjusted to 760 mL by adding purified water, well mixed and transferred to a 1-L volume Erlenmeyer flask. A 240-mL volume of acetonitrile of HPLC grade is measured with the above empty graduated cylinder, and added to the solution in the Erlenmeyer flask. After mixing, the mobile phase solution is passed through a filter (Millicup-HV, Millipore, Bedford, MA, USA) g under reduced pressure. The filtrate is stored in a clean glass container with a stopper at room temperature. A required amount of the above solution is transferred to another glass container, and degassed by immersing the container in an ultrasonic cleaner for 10 min just before use. • 10 mM Phosphate buffer solution (pH 2.1)–10 % acetonitrile (100 mL): a 20-mL volume of 50 mM phosphate buffer solution (pH 2.1) and 10 mL acetonitrile are placed in a graduated cylinder, and the volume of the solution is adjusted with purified water to 100 mL and mixed well. • Pretreatment of a Sep-Pak C18 cartridge b: a 5 mL of methanol, 3 mL of 10 mM phosphate buffer (pH 2.1)–10 % acetonitrile and 5 mL purified water are successively passed through a Sep-Pak C18 cartridge (Waters, Milford, MA, USA) to activate the cartridge just before use. For the handling method, see “Reagents and their preparation”, of the GC and GC/MS analysis section of this chapter. Analytical conditions HPLC column; many analytical methods using reversed phase C18 columns were reported; an example is: a Nova-Pak C18 cartridge column equipped with an RCM × 10 pressurized module; precolumn: Nova-Pak C18 Guard-pak Insert. HPLC conditions; instrument: a Hitachi 655 HPLC instrument (Hitachi Ltd., Tokyo, Japan) equipped a wavelength-variable UV monitor (Hitachi 655A); mobile phase: 5 mM-SDS con- taining 100 mM phosphate buffer (pH 2.1)/acetonitrile (76:24, v/v); flow rate: 1.2 mL/min; detection wavelength: 220 nm.
  9. Toxic and fatal concentrations 203 Procedure A modification of the method developed by Svensson et al. [11] is shown below. i. A 5-mL volume of 0.5 M ammonium sulfate solution (pH 9.4) is added to 2 mL blood plasma or 4 mL of 2-fold diluted urine. When required, the mixture solution is passed through a filter (Cellulose Acetate 0.45 µm, Advantec, Tokyo, Japan). A blank urine speci- men obtained from a healthy subject and the urine specimens spiked with 0.1 mL each of the standard solutions (2, 6 and 20 µg/mL) are also handled in the same way as above. ii. Each of the above solution is passed through a pretreated Sep-Pak C18 cartridge. iii. The cartridge is washed with 20 mL of 5 mM ammonium sulfate solution (pH 9.4) and 0.5 mL purified water. iv. Opiate compounds (free and conjugated forms) trapped in the cartridge are eluted with 1.2 mL methanol. v. The eluate is evaporated to dryness under a stream of nitrogen with warming at 40 °C. The residue is dissolved in 200 µL of the HPLC mobile phase; a 20-µL aliquot of it is injected into HPLC. Assessment and some comments on the method Qualitative analysis is made by the comparison of the retention time of a peak of a specimen with that of the authentic standard and also by the confirmation of the absence of such a peak in a blank specimen. An HPLC chromatogram for the authentic compounds is shown in > Fig. 3.4. For quantitation, the peak area or height of a test compound in a specimen is applied to an ex- ternal calibration curve to calculate its concentration. The use of IS is preferable to make accu- rate quantitation, but the authors have not studied on it. Ethylmorphine may be usable as IS like in the case of GC/MS analysis. With a UV detector, the good linearity of morphine could be confirmed in a range not lower than 20 ng/mL; the detection limit was reported to be 5 ng/mL [11]. With an electrochemical detector, the analysis of opiates is reported to be achievable with even higher sensitivity (detection limit less than about 1 ng/mL) [12]. However, since the elec- trochemical detection is based on the oxidoreductive potential of the hydroxyl group of the phenol moiety, it is not suitable for analysis codeine or M-3-G. For cautions and cutoff values upon detection of trace levels of morphine, the readers can see the 2-4) section of this chapter. By LC/MS, morphine and 6-acetylmorphine give the base peaks of [M+H]+ ions; M-3-G and M-6-G also give [M+H]+ ions (with major fragment ions due to subtraction of glucuronic acid from the molecules) [13, 14]. The quantitativeness in the SIM mode was confirmed; the linearity could be obtained in a range over 30 ng/mL, and the detection limit was 1–3 ng/mL [13]. Toxic and fatal concentrations Fatal doses of morphine and heroin (in the form of their hydrogen chloride salts, per os) were reported to be 70–500 mg and 10–600 mg, respectively [15], with great variation according to individuals. As one of the reasons for the increase in fatal doses, the resistance to an opiate acquired by its repeated use can be mentioned. In a chronic opiate-dependent patient, several grams of an opiate do occasionally not cause death.
  10. 204 Morphine and its analogues ⊡ Figure 3.4 HPLC chromatogram (detected at 220 nm) of the authentic morphine and its related compounds. A: morphine-6-sulfate; B: normorphine-3-glucuronide; C: morphine-3-glucuronide; D: morphine- 6-glucuronide; E: codeine glucuronide; F: morphine. The opiates and their conjugated forms, 10 µg each, were dissolved in 4 mL purified solution, and analyzed according to the procedure described in the section 3-3). As a result of analysis of 203 fatal cases with morphine and heroin, three parameters were reported to be related to the fatality; they are a blood concentration of free morphine (not lower than 0.24 µg/mL), a ratio of the free form to the total morphine (free plus conjugated forms) in blood (not lower than 37 %) and a total morphine concentration in the brain (not lower than 0.08 µg/g and/or higher than the concentration of the free form in blood) [16]. In sudden deaths taking place within 3 h after morphine intake, a phenomenon that the ratio of free morphine to its total amount in blood is not lower than 44 % is said to be characteristically observed [16]. Notes a) Nalorphine at the same concentration can be also used as IS. In the authors’ experience, it is less stable and its solution should be prepared just before use. If a deuterium-labeled morphine is available for use, it is most desirable for MS analysis. b) Convenient thick glass-made devices for assisting elution of extraction cartridges are com- mercially available (GL-SPE Vacuum Manifold, GL Sciences, Tokyo, Japan and some man- ufacturers in USA); it enables the attachment of 12 cartridges, and the simultaneous wash- ing, elution and drying of the cartridges can be made under reduced pressure.
  11. Toxic and fatal concentrations 205 c) When the specimen is blood plasma (1.5 mL), the same volume of 20 % trichloroacetic acid is added, mixed well and centrifuged at 3,000 rpm for 5 min; the resulting supernatant fraction is subjected to be same procedure as that for urine [17, 18]. A solid organ tissue (1 g) is homogenized with the same volume of purified water; the homogenate is deprotei- nized as in the case of the above blood plasma and processed in the same way [18]. The main metabolite M-3-G is not easily hydrolyzed; it requires heating at 100 °C for 30 min in the presence of 15 % hydrochloric acid. The free morphine is relatively unstable and sensi- tive to oxidative decomposition. To protect free morphine from its decomposition, 0.1 mL of 40 % sodium hydrosulfite can be added at the hydrolysis [17, 18]. d) Since in the next derivatization step the reaction is made in a smaller volume, the organic phase filtrate is collected in a 10-mL volume glass centrifuge tube with a ground-in stopper. In this case, the filtration step is divided into several times; about 5 mL of the first filtrate in the 10-mL volume centrifuge tube is subjected to evaporation to reduce its volume, followed by the addition of the next filtrate. This procedure is repeated several times. Upon repeated filtration, it is not necessary to change the paper filter. e) Any BSA reagent commercially available can be used. N,O-Bis(trimethylsilyl)trifluoroacet amide (BSTFA)/trimethylchlorosilane (TMCS) (99:1) can be also used, but no difference in reactivity from the use of BSA can be found. Trifluoroacetic anhydride or propionic anhydride can be used for acylation of opiates. However, the acylating reaction sometimes gives the mixture of monoacyl and diacyl derivatives. f) Since codeine contains a methylated hydroxyl group in the 3-position and thus is not a phenolic base, it is extractable from strongly basic solution. Utilizing this property, it is possible to remove codeine from an extract [17, 19]. Concretely, an organic solvent extract, containing opiates such as morphine and codeine, is back-extracted with 1 M HCl solu- tion; after the pH of the aqueous phase is brought to 12, the phase is extracted with chloro- form, resulting in the extraction of codeine only. g) The filtrate can be collected in a Erlenmeyer flask or a cleaned recycled gallon bottle. When a Erlenmeyer flask is used, the diameter of the mouth of the flask should not be close to that of Millicup-HV, because there is a possibility of destruction of the glassware due to remark- ably reduced pressure. References 1) Fujimoto JM, Way EL (1957) Isolation and crystallization of “blood” morphine from urine of human addicts. J Pharmacol Exp Ther 121:340–346 2) Yoshimura H, Tsukamoto H, Oguri K (1969) Metabolism of drugs-LXII. Isolation and identification of morphine glucuronides in urine and bile of rabbits. Biochem Pharmacol 18:279–286 3) Yoshimura H, Mori M, Oguri K et al. (1970) Metabolism of drugs-LXV. Studies on the urinary conjugated me- tabolites of codeine. Biochem Pharmacol 19:2353–2360 4) Cone EJ, Welch P, Mitchell JM et al. (1991) Forensic drug testing for opiates: I. Detection of 6-acetylmorphine in urine as an indicator of recent heroin exposure, drug and assay considerations and detection times. J Anal Toxicol 15:1–7 5) Ohshita T (1993) Determination of diacetylmorphine in human urine using capillary gas chromatography/mass spectrometry. Eisei Kagaku 39:431–436 (in Japanese with an English abstract) 6) Fehn J, Megges G (1985) Detection of O6-monoacetylmorphine in urine samples by GC/MS as evidence for heroin use. J Anal Toxicol 9:134–138 7) Stefano GB, Goumon Y, Casares F et al. (2000) Endogenous morphine. Trends Neurosci 23:436–442
  12. 206 Morphine and its analogues 8) Matsubara K, Fukushima S, Akane A et al. (1992) Increased urinary morphine, codeine and tetrahydropapavero- line in Parkinsonian patient undergoing L-3,4-dihydroxyphenylalanine therapy: a possible biosynthetic path- way of morphine from L-3,4-dihydroxyphenylalanine in humans. J Pharmacol Exp Ther 260:974–978 9) ElSohly HN, ElSohly MA, Stanford DF (1990) Poppy seed ingestion and opiates urinalysis: a closer look. J Anal Toxicol 14:308–310 10) Hasegawa M, Maseda C, Kagawa M et al. (1992) Morphine and codeine being contained in poppy seeds and poppy seed-containing foods. Eisei Kagaku 38:192–195 (in Japanese with an English abstract) 11) Svensson J-O, Rane A, Säwe J et al. (1982) Determination of morphine, morphine-3-glucuronide and (tenta- tively) morphine-6-glucuronide in plasma and urine using ion-pair high-performance liquid chromatography. J Chromatogr 230:427–432 12) Mason JL, Ashmore SP, Aitkenhead AR (1991) Simple method for the determination of morphine and its active glucuronide metabolite in human plasma by high-performance liquid chromatography with electrochemical detection. J Chromatogr 570:191–197 13) Nishikawa M, Nakajima K, Igarashi K et al. (1992) Analysis of morphine-3-glucuronide in urine by LC/APCI-MS. Eisei Kagaku 38:121–126 (in Japanese with an English abstract) 14) Tatsuno M, Nishikawa M, Katagi M et al. (1996) Simultaneous determination of illicit drugs in human urine by liquid chromatography-mass spectrometry. J Anal Toxicol 20:281–286 15) Nagano T, Wakasugi C (eds) (1995) Modern Legal Medicine, 3rd edn. Kanehara-shuppan, Tokyo, pp 181–183 (in Japanese) 16) Spiehler VR (1998) Computer-assisted interpretation in forensic toxicology: morphine-involved deaths. J Fo- rensic Sci 34:1104–1115 17) Yoshimura H (ed) (1991) Forensic Chemistry, 2nd edn. Nanzando, Tokyo, pp 155–159 (in Japanese) 18) Spiehler V, Brown R (1987) Uncojugated morphine in blood by radioimmunoassay and gas chromatography/ mass spectrometry. J Forensic Sci 32:906–916 19) The Pharmaceutical Society of Japan (ed) (1992) Standard Methods of Chemical Analysis in Poisoning – With Commentary –, 4th edn. Nanzando, Tokyo, pp 230–234 (in Japanese)

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