Journal of Chromatography, 435 (1988) 259-269 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands CHROM. 20 044 REVIEW PHOSGENE AS A DERIVATIZING REAGENT PRIOR TO GAS AND LIQUID CHROMATOGRAPHY* OLLE GYLLENHAAL* and JdRGEN VESSMAN Analytical Chemistry, AB Hiissle, S-431 83 Miilndal (Sweden) (First received January 19th, 1987; revised manuscript received September 7th, 1987) CONTENTS 1. 2. 3. 4. 5. 6. Introduction . . Molecules that cyclize with phosgene 2. I. Derivatization conditions 2.2. Relative reaction rates in dichloromethane Applications . 3.1. Determination of B-blocking drugs and their metabolites chromatography 3.2. Enantiomer separations by gas chromatography 3.3. Enantiomer separations by liquid chromatography Discussion . Acknowledgements . . Summary . . .......... 259 .......... 261 .......... 262 .......... 262 .......... 263 in biological samples by gas .......... 263 .......... 265 .......... 266 .......... 267 .......... 268 .......... 269 References . . . 269 1. INTRODUCTION The use of phosgene as a small-scale derivatizing agent was first reported only a few years ago. Although widely used for large-scale syntheses, especially in the production of intermediates for polyurethane plastics and fine chemicals, until re- cently phosgene has been used mainly in organic chemistry laboratories. Occasion- ally, analytical chemists have come into contact with phosgene as an impurity in chloroform or dichloromethane. Pure phosgene is a gas that liquefies at 8°C. Small amounts are conveniently handled as solutions in toluene; generally a 20% (2 A4) solution can be obtained. It is a noxious compound that should be handled with caution, especially as its odour is not noticeable before toxic concentrations have been reached. It is hydrolysed by water but its reactivity with amines is sufficiently high to allow the formation of derivatives also in an aqueous environment. Many of its reactions are similar to those of acid chlorides and chloroformates. However, a unique property is its ability to l Parts of this paper were presented at the 16th International Symposium on Chromatography, Paris, September 1986. The majority of the papers presented at this symposium have been published in J. Chro- matogr.. Vol. 395 (1987). 0021-9673/88/$03.50 0 1988 Elsevier Science Publishers B.V
Transcript
Journal of Chromatography, 435 (1988) 259-269 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
CHROM. 20 044
REVIEW
PHOSGENE AS A DERIVATIZING REAGENT PRIOR TO GAS AND LIQUID CHROMATOGRAPHY*
OLLE GYLLENHAAL* and JdRGEN VESSMAN
Analytical Chemistry, AB Hiissle, S-431 83 Miilndal (Sweden)
(First received January 19th, 1987; revised manuscript received September 7th, 1987)
CONTENTS
1. 2.
3.
4. 5. 6.
Introduction . . Molecules that cyclize with phosgene 2. I. Derivatization conditions 2.2. Relative reaction rates in dichloromethane Applications . 3.1. Determination of B-blocking drugs and their metabolites
chromatography 3.2. Enantiomer separations by gas chromatography 3.3. Enantiomer separations by liquid chromatography Discussion . Acknowledgements . . Summary . .
.......... 259
.......... 261
.......... 262
.......... 262
.......... 263 in biological samples by gas .......... 263 .......... 265 .......... 266 .......... 267 .......... 268 .......... 269
References . . . 269
1. INTRODUCTION
The use of phosgene as a small-scale derivatizing agent was first reported only a few years ago. Although widely used for large-scale syntheses, especially in the production of intermediates for polyurethane plastics and fine chemicals, until re- cently phosgene has been used mainly in organic chemistry laboratories. Occasion- ally, analytical chemists have come into contact with phosgene as an impurity in chloroform or dichloromethane.
Pure phosgene is a gas that liquefies at 8°C. Small amounts are conveniently handled as solutions in toluene; generally a 20% (2 A4) solution can be obtained. It is a noxious compound that should be handled with caution, especially as its odour is not noticeable before toxic concentrations have been reached. It is hydrolysed by water but its reactivity with amines is sufficiently high to allow the formation of derivatives also in an aqueous environment. Many of its reactions are similar to those of acid chlorides and chloroformates. However, a unique property is its ability to
l Parts of this paper were presented at the 16th International Symposium on Chromatography, Paris, September 1986. The majority of the papers presented at this symposium have been published in J. Chro- matogr.. Vol. 395 (1987).
form cyclic derivatives with bifunctional compounds having the two functional groups in close proximity to each other’.
Derivatization is a standard procedure for preparing compounds that are suf- ficiently volatile or stable to be amenable to gas chromatographic analysis. Certain reactions are also used to improve the detectability of the compound whereas other methods use an inherent property of the molecule for selective and sensitive detection. Derivatization prior to liquid chromatographic analysis has gained popularity, al- though an advantage of liquid over gas chromatography is that labour-intensive derivatization steps are generally not required. The purpose of derivatization in liquid chromatography is often to improve the detection.
Reports of the derivatization of bifunctional compounds, such as amino al- cohols, with phosgene to give cyclic derivatives first appeared in the early 1980s. This type of reaction is expected to give an improved selectivity compared with acylation, alkylation or silylation reactions, as extraneous monofunctional compounds are not likely to react to the same extent and/or show poor gas chromatographic character- istics after derivatization.
This review surveys analytical applications using phosgene as a derivatizing agent prior to chromatography, followed by a discussion on the advantages and disadvantages of using phosgene as a reagent.
2. MOLECULES THAT CYCLIZE WITH PHOSGENE
Examples of structures that form cyclic derivatives are summarized in Table 1. They are briefly discussed in this section, and further details concerning their derivatization and chromatographic analysis are described in later sections.
2-Amino alcohols, such as metoprolol, cyclize with ease to oxazolidin-Zones (oxazolidones). Six- and seven-membered rings can also be formed from correspond- ing 3- and 4-amino alcohols2. Even if the amine and the alcohol groups are part of a ring structure and positioned c( and /?, a cyclized carbamate is formed, as was shown with Organon 6001 as a model compound3. The configuration of the ring makes the distance between the amine and the alcohol closer than the formula would suggest.
When the functional group adjacent to the amine is a carbonyl, a cyclic de- rivative can also be formed, presumably because the ketone is in equilibrium with its enolic form, as found with ketamine 3. Tocainide, a 2_aminopropanamide, gives a hydantoin derivative . 5,6 Amines react and the resulting product cyclizes with aro- matic amines, such as in desethylchloroquine3 (Table 1).
Phenols also react with phosgene. With the catechol moiety of epinephrine a cyclic carbonate derivative was formed. However, owing to its susceptibility to hy- drolysis this derivative was not suitable for quantitative chromatographic analysis7. The hydrolysis of the cyclic carbonate was complete within 1 h at pH 6.5, exposing the catechol group to oxidation, which defeats the purpose of preparing the derivative as early as possible in the analytical procedure in order to protect the catecholamines against oxidation. The overall reaction scheme is shown in Fig. 1. Simple phenolic compounds can be analysed after derivatization with phosgene by the simultaneous addition of methanol to the reaction mixture to form a methyl carbonates.
The aqueous alkaline conditions normally used for derivatization with phos- gene leave isolated functional groups such as hydroxy and carboxyl groups intactg.
262 0. GYLLENHAAL, J. VESSMAN
HN - R pH 7.4
HO ,-, Q
FH - FH2
HO 0 N-R(aPter
‘co’
Fig. 1. Scheme for the reaction of epinephrine (R = CH3) and norepinephrine (R = H) with phosgene and hydrolysis of the carbonate derivatives formed. A Varian 3700 gas chromatograph equipped with a flame
ionization detector was used for the analysis. The glass column (150 x 0.2 cm I.D.) was filled with 3% OV-17 and maintained at 230°C. Nitrogen was used as the carrier gas at a flow-rate of 30 ml/min. Identity
was confirmed by mass spectrometry (Varian-MAT 44S, 70 eV)‘.
Using anhydrous conditions cyclic derivatives were obtained from 1,2- and 1,3-diols and from cr-hydroxy acidslo. N-Methyl-a-amino acids were derivatized in the pres- ence of a small amount of aqueous base, as also were tartaric acid and some tri- functional amino acids after esterification of the carboxyl groupslo.
2.1. Derivatization conditions Although phosgene is hydrolysed by water, derivatization of amino alcohols,
such as metoprolol, can be conveniently performed either in water alone or in the presence of an organic solvent. A two-phase system was preferred for determining metoprolol in plasma samples as the proteins tended to form a gel by the action of phosgene and the agitation of the tubes when buffered plasma samples were treated directly. Addition of 10 ~1 of 2 M phosgene to the system of organic phase and buffered plasma was sufficient for a constant yield of the derivative over the pH range 7.5-122. Simultaneous derivatization of phenols* and in the presence of carboxy groups9 required alkaline conditions (pH 11.5-12) and repeated additions of reagent gave a higher yield of the desired derivative than when all the reagent was added at once. The reaction appears to be almost instantaneou9, but to reduce the risk of exposure to phosgene one should wait at least 10 min in order to allow the excess phosgene be *destroyed by hydrolysis.
Both dichloromethane’O and diethyl ether l l have been used as organic phases and also hexane containing 20% of dichloromethane to accomplish a suitable ex- traction of the derivative with a minimum of interferences from other compounds and less precipitation of proteins in the interface 2. For anhydrous derivatizations, both dichloromethane and dioxane have been reported as solvents’O. Room temper- ature has been used throughout and this is a distinct practical advantage.
2.2. Relative reaction rates in dichloromethane The relative reaction rates of several 3-aryloxy-substituted 1-alkylamino-Zpro-
panol compounds have been studied in dichloromethane3. The results show that the substituent on the amino group considerably influences the rate, whereas the aro- matic part is of little or no importance. The relative rate is 50 times lower for a tert.-butylamino group than for an isopropylamino group, which in turn is ca. 5 times lower than for n-alkylamino groups3.
PHOSGENE AS A DERIVATIZING REAGENT 263
3. APPLICATIONS
This section reviews published work and also some unpublished work on phos- gene as derivatization agent. It is divided into gas chromatography of j?-blockers and their metabolites in biological fluids, gas chromatographic enantiomer separations and finally liquid chromatographic enantiomer separations.
3.1. Determination of P-blocking drugs and their metabolites in biological samples by gas chromatography
The widespread use of /I-adrenergic receptor blocking drugs in clinical practice has resulted in a wide variety of methods for their determination. For gas chro- matographic analysis perfluoroacylation with an anhydride is the most commonly used technique. Other approaches include the use of substituted boronates and sil- ylating agents and also chromatography of the underivatized drugs. Further details and references can be found in a recent review on monitoring of cardiovascular drugsi4.
The first practical application of the phosgene derivatization reaction in gas chromatographic analysis showed that it could be used to determine 8&800 nmol/l of metoprolol in human plasma* with a packed column and nitrogen-selective de- tection. The precision was 1.5% (n = 8) at the 200 nmol/l level. This simple method compared favourably with a standard electron-capture gas chromatographic method with perfluoroacylation*. A drawback is the relatively large sample volume required (2 ml). The simplicity resulted from the combination of extraction and derivatization into one step before concentration by evaporation of the organic phase. Another advantage of the aqueous alkaline two-phase system was that excess phosgene is hydrolysed and need not be considered. A preliminary application to capillary col- umn gas chromatography (Fig. 4 in ref. 2) making it possible to reduce the sample volume, was also investigated.
An extension of this method includes the main metabolites of metoprolol in human urineg. These include a-hydroxylated metoprolol, 0-demethylmetoprolol and metoprolol acid [cJ, Fig. 1 in ref. 91. The last compound, which is the main metab- olite, is an amino acid and therefore requires prior derivatization in order to be extracted. Isolated hydroxy and carboxy groups were found to be unaffected by phosgene in the aqueous alkaline environment used for the oxazolidone formation. In this instance a buffered urine sample was derivatized with three portions of 2 M phosgene in toluene to give an optimal yield of the derivative. The derivatives formed were then extracted into an organic phase at low pH. The remaining polar groups were blocked by trimethylsilylation prior to capillary column gas chromatography. Chromatographic analysis resolved the peaks of metoprolol, 0-demethylmetoprolol, cr-hydroxymetoprolol and metoprolol acid. A lactic acid metabolite eluted in a crowd- ed region of the chromatogram but could be separated under favourable conditions. The precision of the method was about 5% (n = 6) at the 70 pmol/l level, with a limit of detection in the range 420 pmol/l. The practical purpose was to develop a simultaneous method for metoprolol and its main metabolites in urine in order to establish whether individuals can be classified into poor and rapid hydroxylators of metoprolol.
The low selectivity of flame ionization detection combined with the generality
264 0. GYLLENHAAL, J. VESSMAN
of the silylation step applied to acidic urinary extracts resulted in numerous extra- neous peaks (Fig. 6 in ref. 9) and prompted the development of a more selective and sensitive method. This was accomplished by dividing the derivatives formed into a neutral and an acidic fraction. The neutral fraction was extracted at basic pH, then submitted to capillary gas chromatography with nitrogen-selective detection after silylation’ 5,16. Interest was focused on a glycol metabolite, which could be deter- mined with a precision of 3.6% (n = 8) at the 1 pmol/l level in human urine. The chromatographic background was reduced, as many endogenous acids remained in the aqueous phase. By submitting the sample to mass spectral analysis, the presence of this metabolite in urine from a subject who had been given metoprolol was con- firmed. Likewise, the acid fraction was isolated and analysed by mass spectrometry. The mass spectra of c+hydroxylated metabolites derivatized with phosgene followed by trimethylsilylation all showed prominent and thus diagnostic m/z 336 ions.
Pafenolol (Fig. 2a) is a cardioselective /Sadrenoreceptor antagonist, which is mainly excreted as the unchanged drug. A minor metabolite in human urine was derivatized with phosgene followed by trimethylsilylation. The direct probe electron- impact mass spectrum gives the prominent m/z 336 ion (Fig. 2b) which is also found for hydroxylated metoprolol metabolites derivatized by the same method’ 6. The cor-
la1 OH
FH-CH,-NH-CO-NH-CH (CHJ 2
R
lack 0 50 100 156
lack se lee
Fig. 2. (a) Pafenolol (R= H) and its cc-hydroxy metabolite (R=OH). (b) Direct inlet mass spectrum of phosgene/trimethylsilylated cc-hydroxy pafenolol; electron impact at 70 eV. (c) Chemical ionization spec-
trum of a-hydroxypafenolol. After administration of 3H-labelled pafenolol to healthy volunteers, their urine was collected. The isolation procedure consisted of extraction, preparative reversed-phase liquid chromatography, extraction, phosgene derivatization at pH 12 and finally treatment with bis(trimethyl- silyl)acetamide at 60°C for I h before analysis by direct inlet mass spectrometry (Varian-MAT 44s)“.
PHOSGENE AS A DERIVATIZING REAGENT 265
responding methane chemical ionization mass spectrum (Fig. 2c) exhibits MH+ at m/z 452 and the fragmentation pattern supports the suggested structure of the metab- olite (Fig. 2a). The spectra were consistent with those from a synthetic reference”.
In the above methods other functional groups do not give any real problems during the aqueous alkaline phosgene derivatization reaction itself. A common meta- bolic pathway with aromatic compounds is the formation of phenolic metabolites. The higher reactivity of the phenol group compared with alcohols and carboxylic acids prompted the development of a new approach*. By including methanol in the reaction mixture, the initially formed chloroformate was converted into a methyl carbonate. The precision of the method was excellent, about 3% at the 3 pg/ml level, owing to the use of internal standards with a close structural resemblance. However, the estimated yield of the phenolic metabolite was poor, only 24%. This was found to be due to a competing side-reaction in which the initially formed chlorocarbonyl derivative reacted with unreacted phenol and not with methanol. The symmetrical carbonate thus formed decomposed to the phenol oxazolidone in the hot injector of the gas chromatograph and was detected as such (I$., Fig. 2 in ref. 9).
Application of the method for alprenolol and its 4-hydroxy metabolite in urines to real samples18 revealed an interesting lack of selectivity of the method. It was found that the 0-glucuronide conjugates were also converted into oxazolidones by phosgene, although not to the same extent as the free compounds. Studies18 in- dicated that the reactivity of the chlorocarbonylamine intermediate led to the for- mation. of oxazolidones from the 0-glucuronides. To maintain the selectivity of the method it was suggested that any free alprenolol and its 4-hydroxy metabolite should first be isolated by extraction at pH 10. The remaining conjugates are then treated with glucuronidase prior to the derivatization reaction.
3.2. Enantiomer separations by gas chromatography Most of the substituted 3-isopropylamino-2-propanols are used as racemates
although the S-enantiomer is responsible for the biological effect. For example, for metoprolol the S-enantiomer has a ca. 250 times higher b-blocking activity than the R-enantiomer on the heart of a cat lg An interesting property of chiral oxazolidone . derivatives formed with phosgene is the separation of the enantiomers on chiral sta- tionary phases. The capillary column used was coated with a chiral polysiloxane XE-60-L-valine-(R)-cc-phenylethylamide phase 20. These derivatives are evidently still sufficiently polar to interact with the stationary phase. Oxazolidones of P-blockers are stable both in solution towards hydrolysis and chromatographically towards ac- tive sites in the column, in contrast to the more labile perfluoroacyl derivatives21. Partial inversion of ephedrine into pseudoephedrine with heptafluorobutyric anhy- dride has been observed22. For the first time the separation was also accomplished of N-tert.-butyl-substituted p-blocking drugs such as bupranol and penbutol (Fig. 2 in ref. 20). With the pure enantiomers it was shown that the R-isomers eluted first23. No racemization was observed when the pure isomers were derivatized separately. The volatility of the oxazolidones is lower than that of corresponding HFB deriva- tives. Using an 18-m column at 195°C they elute within 70 min (hydrogen inlet pres- sure 80 kPazo).
A practical enantiomer separation has been demonstrated with metoprolol acid after cyclization with phosgene and methylation with diazomethane24. Analysis of
266 0. GYLLENHAAL, J. VESSMAN
urine collected from a healthy volunteer who had taken a dose of metoprolol revealed no change in the enantiomeric ratio by the metabolic conversion of metoprolol. The degree of separation of enantiomers of metoprolol and related compounds was also studiedz4. The results showed that a five-membered ring (oxazolidone) gave a su- perior separation to a six-membered ring and that the N-substituent is of minor importance for the enantiomer separation. The c+hydroxy metabolite of metoprolol, H 119/66, was separated as its phosgene-TMS derivative. Here a new chiral centre is present but it is assumed that the oxazolidone is responsible for the separation.
Phenolic amino alcohols can be derivatized with methanol present in the aqueous alkaline phosgene system used *,18. A drawback of these methyl carbonate oxazolidone derivatives is their polar nature and long retention times on the chiral column used for the enantiomer separation 23. To overcome this problem the sub- stances were first methylated with diazomethane 24*25. The aromatic methyl ether derivatives thus formed were then treated with phosgene and subjected to gas chro- matography. At 190°C with hydrogen as the carrier gas the recorded cl-values were in the range 1.024 (4-hydroxyalprenolol) to 1.044 (phenylephrine). In Fig. 3 an actual separation is shown for terbutaline, a /?-receptor stimulating drug used in the treat- ment of asthma.
Not only Q- and b-receptor active drugs have been separated after phosgene derivatization. This reagent and the derivatives formed also work well with chiral molecules such as 1,2- and 1,3-diols, cc-hydroxy acids, N-methyl-a-amino acids and mercaptoamino acids’ o*2 3. The enantiomer separation of aliphatic diols was possible for the first time after formation of cyclic carbonates (Fig. 2 in ref. 10).
3.3. Enantiomer separations by liquid chromatography The usefulness of oxazolidones formed from amino alcohols with phosgene for
their enantiomeric separation has been shown with a variety of chromatographic systems such as Pirkle columns’ 1,27, a,-acid glycoprotein28.29, triacetylcellulose30 and modified silica gel 3 1-33 Liquid chromatographic enantiomer separation offers . some advantages over corresponding gas chromatographic separations. Although baseline separation is not so easily achieved it is still possible to chromatograph and
min 15 0
Fig. 3. Gas chromatographic enantiomer separation of terbutaline after methylation with diazomethane and reaction with phosgene. Chromatographic conditions: column, XE-60-L-val-(R)-cc-phenylethylamide, oven temperature 200°C carrier gas hydrogen (100 kPa). Further details are given in refs. 25 and 26.
PHOSGENE AS A DERIVATIZING REAGENT 267
separate enantiomers on a preparative scale 27,30. Regeneration of the oxazolidone formed from (+)-norephedrine was carried out by refluxing for 5 h in 1.5 M potas- sium hydroxide in 50% methanol 27 The hydrolysis proceeded without racemization, . which was shown after a second derivatization with phosgene followed by chiral liquid chromatography2’, which is in line with earlier results34p35.
The separation of the oxazolidones of metoprolol and propranolol on triace- tylcellulose on a lo-mg scale has been reported 30. The superior separation compared with the underivatized molecules was proposed to be due to reduced hydrogen bond- ing between the solute and the solvent. The dipole-dipole interaction between the ester carbonyl of the support and that of the solute may also contribute to the chiral recognition. The possibility of recovering the original compounds after alkaline hy- drolysis was pointed out.
Three optically active supports were prepared from different derivatives of glucose and 1-aminopropyl-silica gel 3 1-33. Oxazolidones of several /?-blocking drugs including penbutolol and toliprolol have also been separated into their enantiomers3’. The mobile phase consisted of n-hexane with 6% of 2-propanol and a-factors about 1.2 on 3,5-dinitrobenzoyltriacetylglucose were reported. The support was prepared from modified 1-isothiocyanato-o-glucopyranosides covalently bound to aminopropyl-silica ge131. The separation of racemic epinephrine (a = 1.45) on triacetyl-2-acetamido-2-deoxyglucose as the chiral selector31 was also reported (Fig. 3 in ref. 33).
Many racemic drugs can be separated into their enantiomers without deriva- tization, using al-acid glycoprotein-coated silica columns. With P-blocking drugs, transformation to oxazolidones results in an increase in retention and a drastic im- provement in chiral resolution 28,2Q. The stereoselectivity is remarkably high con- sidering that the separation is obtained in the presence of 10% of propanol, which in most instances reduces the stereoselectivity 2Q. Under favourable chromatographic conditions the a values were in the range 1.6 (alprenolol) to 5.7 (propranolol)26.
Quantitative enantiomer determination has also been reported’ l. The enan- tiomers of propranolol were isolated from whole blood by diethyl ether extraction and derivatized with phosgene, followed by separation on a Pirkle type 1-A column. Standards were prepared in the range 0.5-100 ng/ml and a fluorescence detector was used to monitor the column effluent. Pronethanol, which is propranolol lacking the oxymethylene bridge between the aromatic ring and the isopropylaminopropanol side-chain, was used as an internal standard. Interestingly, the standard did not sep- arate into its enantiomers. The precision was about 5% (n = 5) at the 50 ng/ml level for both isomers. A blood concentration-time curve was obtained after dosing one subject with 80 mg of racemic propranolol hydrochloride’ l.
4. DISCUSSION
From the previous section, it is obvious that phosgene in many instances is an attractive derivatizing agent prior to chromatographic analysis of especially amino alcohols such as drugs of the /I-blocking type. Most of the hitherto published methods involve compounds of this class. Derivatization with phosgene has been used prior to both gas and liquid chromatographic analysis. The chemical stability of the oxa- zolidone derivatives compares favourably with that of other cyclic derivatives from
268 0. GYLLENHAAL, J. VESSMAN
amino alcohols such as those obtained with substituted boronic acids’ or aldehydes (oxazolidines). The stability of oxazolidines towards hydrolysis is generally 10~~~~~‘. By using strong alkali the amino alcohol can be generated from the oxazolidone derivative*‘. The relative stability is also superior to that of the derivatives obtained with silylating reagents (trimethylsilyl) and perfluoroanhydrides38. The perfluoroacyl derivatives are sensitive to moisture and require deactivation of the capillary column to prevent degradation of the derivatives during the chromatographic step* l. Tri- methylsilyl ethers of alcohols are easily formed and stable whereas amines require potent trimethylsilyl donors and the presence of an excess of the reagent until injec- tion.
The sensitivity of certain detectors does not increase with phosgene derivati- zation, which is the case with perfluoroacyl reagents. By using a nitrogen-selective detector a certain selectivity is obtained from the nitrogen atom already present in the molecule*. Mass-selective detection is another means of improving the selectivity compared with flame ionization detection 16. The strong hydrogen bonding capacity of the amino alcohols is not present in the oxazolidones. However, the molecule is still fairly polar and partitioning into dichloromethane was only marginally better than that for the parent compound*. The superior packed column chromatographic behaviour on the ester phase HiEFF-8BP compared with OV-17 also supports this observation*.
The solution of the reagent in toluene appears to be very pure and no extra- neous peaks emanating from the reagent have so far been reported. In some instances side-reactions occur, e.g., when derivatizing phenols with methanol present*. Studies on the possibility of other amines or phenols competing with the cyclization of the chlorcarbonylamine intermediate* showed that with dimethylamine and phenol this did not occur, except for the n-propyl analogue of metoprolol in the presence of dimethylamine, where a 30% reduction in the relative yield of the derivative was observed3. Hence, the possibility that dimers of P-blocking drugs should be formed at low analytical concentrations (< lo-’ M) appears less likely.
All examples of liquid chromatographic separations of oxazolidones have been aimed at improving the enantiomeric separation in different chromatographic sys- tems. The hydrogen bonding ability is sufficiently reduced and the rigid structure of the cyclic derivative favours the enantioselectivity of the chromatographic column.
The derivatization procedure itself offers several advantages such as working at room temperature, the reagent is consumed and no excess has to be removed and the procedure can be performed in the presence of an alkaline aqueous phase that serves as a proton scavenger. However, the reagent itself has a poor reputation among most chemists, which apparently goes back to the use of phosgene as a war gas during World War I. Nevertheless, it is common practice to work in a fume-hood and the reagent can be dispensed from a stock solution into a small vial for immediate use. In our experience a push-button syringe (Hamilton, Bonaduz, Switzerland) has proved convenient for dispensing the reagent into the sample tubes.
5. ACKNOWLEDGEMENTS
Our thanks are due to Dr. Kurt-Jiirgen Hoffman, Professor Wilfried A. Konig and Professor Bo Lamm for valuable discussions and to Dr. M. Ahnoff for critically reading the manuscript.
PHOSGENE AS A DERIVATIZING REAGENT 269
6. SUMMARY
The use of phosgene as a derivatizing agent for bifunctional compounds prior to gas and liquid chromatographic analysis is reviewed. Applications include gas chromatographic determinations of metoprolol and its metabolites in biological fluids, enantiomeric separations of b-blocking drugs and sympatomimetic agents on a chiral stationary phase and liquid chromatographic enantiomer separations.
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