Acta pharmacol. et toxicol. 1977, 41,273-279.
From the Department of Pharmacology, Faculty of Pharmacy, University of Uppsala, Biomedical Center, Box 573, S-75123 Uppsala, and Apoteksbolaget AB, Box 3045, S-17103 Solna 3, Sweden
Simultaneous Determination of Codeine and Morphine in Biological Samples by Gas Chromatography with Electron Capture Detection BY Bengt Dahlrtrom, Lennart Paalzow and Per Olov Edlund (Received December 21, 1976;Accepted March 2, 1977)
Abstract: A sensitive gas chromatographic method for the simultaneous determination of codeine and morphine in plasma and brain samples is described. The method involves solvent extraction of the compounds from plasma, derivatization with pentafluoropropionic anhydride and subsequent separation on a 3 % OV-17 column. The quantification is performed with electron capture detection. The sensitivity of the method (0.75 ng of morphine and 7.5 ng of codeine in a sample) makes it especially useful for pharmacokinetic investigations, The method was successfully applied to determine the time course of codeine and its metabolite morphine after intravenous administration of codeine to the rat.
-
Key-words: Morphine codeine determination.
-
gas chromatography
-
plasma and brain
In pharmacokinetic and bioavailability investigations there is a need for highly sensitive and specific assays of drugs in tissues, usually in the nanogram range. As many drugs also give rise to pharmacologically active metabolites, it is necessary to quantify these as well, in order to obtain correct studies on, for example, the relation between plasma levels of drugs and the intensity of the pharmacological effect. Many assay methods for codeine and morphine have appeared in the literature (SCHMERZLER et al. 1966; BRUNSON& NASH1975; NAKAMURA & WAY1975; SERFONTEIN et al. 1975;ZWEIDINGER et al. 1976). Most of these methods utilize gas chromatography with flame ionization detection, but due to the sensitivity limit of this detector, rather large sample volumes are necessary to reach sufficient sensitivity. This is a limiting factor when performing pharmacokinetic investigations in small animals.
274
B. DAHLSTROM, L. PAALZOW AND P. 0. EDLUND
However, by using electron capture detection of suitable derivatives of the substances (WALLACE et al. 1974; DAHLSTROM & PAALZOW 1975;KOGAN& CHEDEKEL 1976), it is possible to increase the sensitivity and thus reduce the sample volume necessary for the analysis. A gas chromatographic/mass spectrometric method has been described by EBBIGHAUSEN et al. (1973), but this sophisticated equipment is not yet commonly available. The use of gas chromatography together with electron capture detection of codeine derivatives provides an opportunity to increase the sensitivity and also the possibility of analysing simultaneously small amounts of both codeine and morphine in the same sample. The present article describes a specific and sensitive gas chromatographic method with electron capture detection which is especially suited for simultaneous determination, in the na.nogram/ml range, olf codeine and morphine in biological samples.
Materials and Methods Instruments. The analyses were carried out on Varian Aerographs (made1 1400) equipped with either 'H-Scandium or JH-Titan electron capture detectors. The detectors were operated at 275" and 225O, respectively. The electrometers were connected to an electronic integrator (Spectra-Physics, Autolab. div.) and to Varian mod. 25 recorders. The separations were performed on 1.8 m X 2 mm silanized glass columns packed with 3 % OV-17 on Gas-Chrom Q (100-120 mesh). The column temperature was maintained at 215". The carrier gas (nitrogen) was purified by 2 in-line filters for removal of oxygen traces and water vapour. The gas-flow rate was maintained at 30 ml/min. The injector was operated at 220". The injections (1-2 pl) were made in an oncolumn mode, either with a Varian Autoinjector 8000, readjusted for sample volumes of 50-100 ~ l o,r manually. The columns were regularly treated with 50 pl of Silyl-8 (Pierce Chemical Co.) in order to achieve high column performance. Reagents and Chemicals. Toluene, butanol and ethyl acetate (E.Merck, Darmstadt, G. F. R.) of P. A. quality were purified by fractional distillation before use. Pentafluoropropionic-anhydride (PPPA) was obtained from Produktkontroll AB (Solna, Sweden). Morphine, 3-0-ethylmorphine and codeine were obtained from the WHO Center for Chemical Reference Substanca (Solna, Sweden). N-ethyl-normorphine was synthetized from normorphine mainly according to EBBIOHAUSBN ei al. (1973). The reaction was monitored by TLC. The reaction mixture was extracted with to1uene:butanol (9:1), and after evaporation the solution was subjected to preparative TLC (Silicagel-G with fluorescence indicator, MerckQ, Darmstadt). The appropriate band was scraped off and packed in a column which was eluted with 0.1 M-H,SO,. The eluate was then extracted with to1uene:butanol at pH 8.9. The organic phase was evaporated and the substance redissolved in 10-4 M-HCl. The purity obtained (>99 %) was checked by gas chromatography with electron capture detection. All glassware was carefully silanized by Dri-film or SiIyl-8 (Pierce Chemical Co.).
GLC OF MORPHINE AND CODEINE
275
Procedure.
A. To each plasma sample (0.05-1.0 ml), an appropriate amount of the internal standards (3-ethyl-morphine and N-ethyl-normorphine) was added. The extraction procedure was performed according t o paragraph C below. B. Brain samples were homogenized in 0.4 M-HCIO, (with 0.1 96 sodiumsulphite and 0.05 % disodium EDTA) containing the internal standards. 2.6 ml perchloric acid was used per gram of brain-tissue. The homogenate was centrifuged for 20 min. at 18,000 r.p.m., at 1". An aliquot of the supernatant was then used for the subsequent analysis according to paragraph C below. C. The samples were adjusted to pH 8.9 with carbonate buffer (1.5 M) and 3.0 ml toluene:butanoI (9:l) was added. The tubes were shaken on a Biichler Rotamixer for 30 min. and centrifuged at lo00 r.p.m. for 5 min. The organic phase was transferred to a second tube containing 0.5 ml 0.1 M-H,SO, and shaken for another 15 min. The organic phase was discarded and the acidic water phase shaken once more with 3.0 ml toluene:butanol (9:l). After centrifugation and removal of the organic phase, the aqueous phase was adjusted with carbonate buffer (1.5 M) to pH 8.9. An amount of 3.0 ml to1uene:butanol (9:l) was added and the tubes were shaken for 15 min., and then centrifuged. The organic phase was transferred to silanized reaction vials and evaporated to dryness in a heating block under a gentle stream of nitrogen. 100 p1 of PFPA was added to each reaction vial, which was then placed in an oven or a heating block for 30 min.at 65".
PEAK AREA RATIO
1.0
0.8-
MORPUINE 4INE MPLE
loo mo &o
Mo
CooEINE
Fig. 1. Typical example of a calibration curve for morphine and codeine.
276
B. DAHLSTReM, L. PAALZOW AND P. 0. EDLUND
The excess of reagent was removed by a gentle stream of nitrogen at room temperature and the sample was redissolved in 75-100 pl dry glass-distilled ethyl acetate. 1-2 pl of this solution was injected into the gas chromatograph. The areas under the peaks were calculated by the electronic integrator, using an attenuation of 2 X 10-10 on the electrometer for the input-signal to the integrator. Calibration curves were determined from plots of area-ratios against known concentrations of the two substances, as exemplified in fig. 1.
Results and Discussion
A solvent extraction technique was employed to provide the necessary clean-up of the plasma and brain samples. The organic solvent [toluene: butanol (9:1)] was chosen because it gives very clean blank extracts from plasma and brain and, furthermore, the addition of butanol prevents the adsorption of the alkaloids to the glass ware (DAHLSTROM & PAALZOW 1975). By adjusting the pH to 8.9, it was possible to extract both morphine and codeine and their respective internal standards simultaneously to the organic phase. The inclusion of an extra extraction step of the acidic water-phase further reduced the “background” noise, as compared to the previously published analytical method for morphine (DAHLSTROM & PAALZOW 1975). The recovery of materia1 across the extraction procedure was 67 f 4 % for morphine, and 89 J- 7% for codeine (mean k S.E.M., n = 6). The internal standards 3-0-ethyI-morphine and N-ethyl-normorphine were chosen because of their close chemical relationship to codeine and morphine, respectively. Furthermore, it is advantageous that the internal standards have the same functional groups that will react with the PFPA as the corresponding parent compound i.e. morphine and codeine. The inclusion of an extra carbon atom, compared to the parent substances, gave the internal standards the prolonged retention time necessary to obtain a good separation of the peaks (fig. 2). The use of two internal standards was necessary to obtain a low coefficient of variation, especially for morphine. Nalorphine, which was used in the previously reported assay of morphine (DAHLSTROM & PAALZOW 1975), could not be used on the 3 % OV-17 column due to interference with the codeine peak. The reproducibilities of the method for 2 different sample quantities are given in table 1. PFPA was chosen as derivatizing agent since it gives the derivatives good electron capture properties. Furthermore, the derivatization can be camed out in one step for both morphine and codeine, and also the removal of the excess of reagent is very convenient due to its volatility. The sensitivity of the method allowed detmminations of codeine and
277
GLC O F MORPHINE AND CODEINE
IF FULL SCALE IESPONSE
e I
e
a
e
s
10
ie
14 ie MINUTES
Fig. 2. A chromatogram of morphine and codeine extracted from plasma. The concentrations of morphine and codeine were 40 ng/ml and 400 ng/ml, respectively. 1 represents morphine, 2 N-ethyl-normorphine, 3 codeine, and 4 3-0-ethyl-morphine.
morphine with acceptable precision down to 7.5 ng and 0.75 ng, respectively, in each sample. Morphine and its internal standard N-ethyl-normorphine have hydroxyl groups in positions 3 and 6 and these groups reacted with the PFPA to yield the corresponding diester with the derivatizing agent. Codeine and its internal standard 3-0-ethyl-morphine have one hydroxyl group in position 6 which can react with the PFPA to yield the corresponding monoester derivatives. Table 1. Reproducibility of multiple determinations of morphine and codeine. N = Number of analyses. Amount present in sample Morphine Codeine
S.D. %
N
19 ng 5 ng
3.7 6.6
7 7
215 ng 43 ng
0.5 4.9
7 13
278
B. DAHLSTRCiM, L. PAALZOW AND P. 0. EDLUND
LN PLASMA CONC. INQ/ML I
2
s R " o - ;
Fig. 3. Time course of plasma concentrations of codeine and morphine after an intravenous injection of 20 mg/kg of codeine phosphate to a rat.
The structures of the derivatives were confirmed by mass spectrometry. The usefulness of the method in pharmacokinetic studies was investigated by monitoring plasma levels of codeine and morphine after intravenous administration of codeine phosphate (30 mgkg) into rats. Because of the small sample volumes necessary for quantification, it was possible to follow the whole concentration time profile of both codeine and morphine simultaneously in each animal (fig. 3).
REFERENCES Brunson, M. K. & J. F. Nash: Gaschromatographic measurement of codeine and norcodeine in human plasma. Clin. Chem. 1975,21, 1965-1960. Dahlstrom, B. & L. Paalzow: Quantitative determination of morphine in biological samples by gas-liquid chromatography and electron capture detection. J . Pharm. Pharmacol. 1975, 27, 172-176. Ebbighausen, W. 0. R., J. H. Mowat, P. Vestergaard & N. S. Kline: Stable isotope method for the assay of codeine and morphine by gas chromatography-mass spectrometry. A feasibility study. Adv. Biochem. Psychopharmacol. 1973, 7 , 135146. Kogan, M. J. & M. A. Chedekel: Rapid g.1.c. method for the separation of picogram quantities of morphine and codeine. J . Pharm. Pharniacol. 1976, 28, 261. Nakamura, G. R. & E. L. Way: Determination of morphine and codeine in postmortem specimens. Anal, Chem. 1975, 47,775-778. Schmenler, E., W. Yu, M. 1. Hewitt & I. J. Greenblatt: Gas chromatographic determination of codeine in serum and urine. J . Pharrn. Sci. 1966, 55, 155-157.
GLC OF MORPHINE AND CODEINE
279
Serfontein, W. J., D. Botha & L. de Villiers: A rapid GLC procedure for the determination of codeine and norcodeine in biological fluids based on microphase extraction techniques. J . Pharm. PharmacoI. 1975,27,937-939. Zweidinger, R. A., F. M. Weinberg & R. W. Handy: Quantitative determination of codeine in plasma. J. Pharm. Sci. 1976,65,427429. Wallace, J. E., H. E. Hamilton, K. Blum & C. Petty: Determination of morphine in biologic fluids by electron capture gas-liquid chromatography. AnaZ. Chem. 1974, 46, 21W-2110.
From the Department of Pharmacology, Faculty of Pharmacy, University of Uppsala, Biomedical Center, Box 573, S-75123 Uppsala, and Apoteksbolaget AB, Box 3045, S-17103 Solna 3, Sweden
Simultaneous Determination of Codeine and Morphine in Biological Samples by Gas Chromatography with Electron Capture Detection BY Bengt Dahlrtrom, Lennart Paalzow and Per Olov Edlund (Received December 21, 1976;Accepted March 2, 1977)
Abstract: A sensitive gas chromatographic method for the simultaneous determination of codeine and morphine in plasma and brain samples is described. The method involves solvent extraction of the compounds from plasma, derivatization with pentafluoropropionic anhydride and subsequent separation on a 3 % OV-17 column. The quantification is performed with electron capture detection. The sensitivity of the method (0.75 ng of morphine and 7.5 ng of codeine in a sample) makes it especially useful for pharmacokinetic investigations, The method was successfully applied to determine the time course of codeine and its metabolite morphine after intravenous administration of codeine to the rat.
-
Key-words: Morphine codeine determination.
-
gas chromatography
-
plasma and brain
In pharmacokinetic and bioavailability investigations there is a need for highly sensitive and specific assays of drugs in tissues, usually in the nanogram range. As many drugs also give rise to pharmacologically active metabolites, it is necessary to quantify these as well, in order to obtain correct studies on, for example, the relation between plasma levels of drugs and the intensity of the pharmacological effect. Many assay methods for codeine and morphine have appeared in the literature (SCHMERZLER et al. 1966; BRUNSON& NASH1975; NAKAMURA & WAY1975; SERFONTEIN et al. 1975;ZWEIDINGER et al. 1976). Most of these methods utilize gas chromatography with flame ionization detection, but due to the sensitivity limit of this detector, rather large sample volumes are necessary to reach sufficient sensitivity. This is a limiting factor when performing pharmacokinetic investigations in small animals.
274
B. DAHLSTROM, L. PAALZOW AND P. 0. EDLUND
However, by using electron capture detection of suitable derivatives of the substances (WALLACE et al. 1974; DAHLSTROM & PAALZOW 1975;KOGAN& CHEDEKEL 1976), it is possible to increase the sensitivity and thus reduce the sample volume necessary for the analysis. A gas chromatographic/mass spectrometric method has been described by EBBIGHAUSEN et al. (1973), but this sophisticated equipment is not yet commonly available. The use of gas chromatography together with electron capture detection of codeine derivatives provides an opportunity to increase the sensitivity and also the possibility of analysing simultaneously small amounts of both codeine and morphine in the same sample. The present article describes a specific and sensitive gas chromatographic method with electron capture detection which is especially suited for simultaneous determination, in the na.nogram/ml range, olf codeine and morphine in biological samples.
Materials and Methods Instruments. The analyses were carried out on Varian Aerographs (made1 1400) equipped with either 'H-Scandium or JH-Titan electron capture detectors. The detectors were operated at 275" and 225O, respectively. The electrometers were connected to an electronic integrator (Spectra-Physics, Autolab. div.) and to Varian mod. 25 recorders. The separations were performed on 1.8 m X 2 mm silanized glass columns packed with 3 % OV-17 on Gas-Chrom Q (100-120 mesh). The column temperature was maintained at 215". The carrier gas (nitrogen) was purified by 2 in-line filters for removal of oxygen traces and water vapour. The gas-flow rate was maintained at 30 ml/min. The injector was operated at 220". The injections (1-2 pl) were made in an oncolumn mode, either with a Varian Autoinjector 8000, readjusted for sample volumes of 50-100 ~ l o,r manually. The columns were regularly treated with 50 pl of Silyl-8 (Pierce Chemical Co.) in order to achieve high column performance. Reagents and Chemicals. Toluene, butanol and ethyl acetate (E.Merck, Darmstadt, G. F. R.) of P. A. quality were purified by fractional distillation before use. Pentafluoropropionic-anhydride (PPPA) was obtained from Produktkontroll AB (Solna, Sweden). Morphine, 3-0-ethylmorphine and codeine were obtained from the WHO Center for Chemical Reference Substanca (Solna, Sweden). N-ethyl-normorphine was synthetized from normorphine mainly according to EBBIOHAUSBN ei al. (1973). The reaction was monitored by TLC. The reaction mixture was extracted with to1uene:butanol (9:1), and after evaporation the solution was subjected to preparative TLC (Silicagel-G with fluorescence indicator, MerckQ, Darmstadt). The appropriate band was scraped off and packed in a column which was eluted with 0.1 M-H,SO,. The eluate was then extracted with to1uene:butanol at pH 8.9. The organic phase was evaporated and the substance redissolved in 10-4 M-HCl. The purity obtained (>99 %) was checked by gas chromatography with electron capture detection. All glassware was carefully silanized by Dri-film or SiIyl-8 (Pierce Chemical Co.).
GLC OF MORPHINE AND CODEINE
275
Procedure.
A. To each plasma sample (0.05-1.0 ml), an appropriate amount of the internal standards (3-ethyl-morphine and N-ethyl-normorphine) was added. The extraction procedure was performed according t o paragraph C below. B. Brain samples were homogenized in 0.4 M-HCIO, (with 0.1 96 sodiumsulphite and 0.05 % disodium EDTA) containing the internal standards. 2.6 ml perchloric acid was used per gram of brain-tissue. The homogenate was centrifuged for 20 min. at 18,000 r.p.m., at 1". An aliquot of the supernatant was then used for the subsequent analysis according to paragraph C below. C. The samples were adjusted to pH 8.9 with carbonate buffer (1.5 M) and 3.0 ml toluene:butanoI (9:l) was added. The tubes were shaken on a Biichler Rotamixer for 30 min. and centrifuged at lo00 r.p.m. for 5 min. The organic phase was transferred to a second tube containing 0.5 ml 0.1 M-H,SO, and shaken for another 15 min. The organic phase was discarded and the acidic water phase shaken once more with 3.0 ml toluene:butanol (9:l). After centrifugation and removal of the organic phase, the aqueous phase was adjusted with carbonate buffer (1.5 M) to pH 8.9. An amount of 3.0 ml to1uene:butanol (9:l) was added and the tubes were shaken for 15 min., and then centrifuged. The organic phase was transferred to silanized reaction vials and evaporated to dryness in a heating block under a gentle stream of nitrogen. 100 p1 of PFPA was added to each reaction vial, which was then placed in an oven or a heating block for 30 min.at 65".
PEAK AREA RATIO
1.0
0.8-
MORPUINE 4INE MPLE
loo mo &o
Mo
CooEINE
Fig. 1. Typical example of a calibration curve for morphine and codeine.
276
B. DAHLSTReM, L. PAALZOW AND P. 0. EDLUND
The excess of reagent was removed by a gentle stream of nitrogen at room temperature and the sample was redissolved in 75-100 pl dry glass-distilled ethyl acetate. 1-2 pl of this solution was injected into the gas chromatograph. The areas under the peaks were calculated by the electronic integrator, using an attenuation of 2 X 10-10 on the electrometer for the input-signal to the integrator. Calibration curves were determined from plots of area-ratios against known concentrations of the two substances, as exemplified in fig. 1.
Results and Discussion
A solvent extraction technique was employed to provide the necessary clean-up of the plasma and brain samples. The organic solvent [toluene: butanol (9:1)] was chosen because it gives very clean blank extracts from plasma and brain and, furthermore, the addition of butanol prevents the adsorption of the alkaloids to the glass ware (DAHLSTROM & PAALZOW 1975). By adjusting the pH to 8.9, it was possible to extract both morphine and codeine and their respective internal standards simultaneously to the organic phase. The inclusion of an extra extraction step of the acidic water-phase further reduced the “background” noise, as compared to the previously published analytical method for morphine (DAHLSTROM & PAALZOW 1975). The recovery of materia1 across the extraction procedure was 67 f 4 % for morphine, and 89 J- 7% for codeine (mean k S.E.M., n = 6). The internal standards 3-0-ethyI-morphine and N-ethyl-normorphine were chosen because of their close chemical relationship to codeine and morphine, respectively. Furthermore, it is advantageous that the internal standards have the same functional groups that will react with the PFPA as the corresponding parent compound i.e. morphine and codeine. The inclusion of an extra carbon atom, compared to the parent substances, gave the internal standards the prolonged retention time necessary to obtain a good separation of the peaks (fig. 2). The use of two internal standards was necessary to obtain a low coefficient of variation, especially for morphine. Nalorphine, which was used in the previously reported assay of morphine (DAHLSTROM & PAALZOW 1975), could not be used on the 3 % OV-17 column due to interference with the codeine peak. The reproducibilities of the method for 2 different sample quantities are given in table 1. PFPA was chosen as derivatizing agent since it gives the derivatives good electron capture properties. Furthermore, the derivatization can be camed out in one step for both morphine and codeine, and also the removal of the excess of reagent is very convenient due to its volatility. The sensitivity of the method allowed detmminations of codeine and
277
GLC O F MORPHINE AND CODEINE
IF FULL SCALE IESPONSE
e I
e
a
e
s
10
ie
14 ie MINUTES
Fig. 2. A chromatogram of morphine and codeine extracted from plasma. The concentrations of morphine and codeine were 40 ng/ml and 400 ng/ml, respectively. 1 represents morphine, 2 N-ethyl-normorphine, 3 codeine, and 4 3-0-ethyl-morphine.
morphine with acceptable precision down to 7.5 ng and 0.75 ng, respectively, in each sample. Morphine and its internal standard N-ethyl-normorphine have hydroxyl groups in positions 3 and 6 and these groups reacted with the PFPA to yield the corresponding diester with the derivatizing agent. Codeine and its internal standard 3-0-ethyl-morphine have one hydroxyl group in position 6 which can react with the PFPA to yield the corresponding monoester derivatives. Table 1. Reproducibility of multiple determinations of morphine and codeine. N = Number of analyses. Amount present in sample Morphine Codeine
S.D. %
N
19 ng 5 ng
3.7 6.6
7 7
215 ng 43 ng
0.5 4.9
7 13
278
B. DAHLSTRCiM, L. PAALZOW AND P. 0. EDLUND
LN PLASMA CONC. INQ/ML I
2
s R " o - ;
Fig. 3. Time course of plasma concentrations of codeine and morphine after an intravenous injection of 20 mg/kg of codeine phosphate to a rat.
The structures of the derivatives were confirmed by mass spectrometry. The usefulness of the method in pharmacokinetic studies was investigated by monitoring plasma levels of codeine and morphine after intravenous administration of codeine phosphate (30 mgkg) into rats. Because of the small sample volumes necessary for quantification, it was possible to follow the whole concentration time profile of both codeine and morphine simultaneously in each animal (fig. 3).
REFERENCES Brunson, M. K. & J. F. Nash: Gaschromatographic measurement of codeine and norcodeine in human plasma. Clin. Chem. 1975,21, 1965-1960. Dahlstrom, B. & L. Paalzow: Quantitative determination of morphine in biological samples by gas-liquid chromatography and electron capture detection. J . Pharm. Pharmacol. 1975, 27, 172-176. Ebbighausen, W. 0. R., J. H. Mowat, P. Vestergaard & N. S. Kline: Stable isotope method for the assay of codeine and morphine by gas chromatography-mass spectrometry. A feasibility study. Adv. Biochem. Psychopharmacol. 1973, 7 , 135146. Kogan, M. J. & M. A. Chedekel: Rapid g.1.c. method for the separation of picogram quantities of morphine and codeine. J . Pharm. Pharniacol. 1976, 28, 261. Nakamura, G. R. & E. L. Way: Determination of morphine and codeine in postmortem specimens. Anal, Chem. 1975, 47,775-778. Schmenler, E., W. Yu, M. 1. Hewitt & I. J. Greenblatt: Gas chromatographic determination of codeine in serum and urine. J . Pharrn. Sci. 1966, 55, 155-157.
GLC OF MORPHINE AND CODEINE
279
Serfontein, W. J., D. Botha & L. de Villiers: A rapid GLC procedure for the determination of codeine and norcodeine in biological fluids based on microphase extraction techniques. J . Pharm. PharmacoI. 1975,27,937-939. Zweidinger, R. A., F. M. Weinberg & R. W. Handy: Quantitative determination of codeine in plasma. J. Pharm. Sci. 1976,65,427429. Wallace, J. E., H. E. Hamilton, K. Blum & C. Petty: Determination of morphine in biologic fluids by electron capture gas-liquid chromatography. AnaZ. Chem. 1974, 46, 21W-2110.
