443
Clinica Chimica Acta, 62 (1975) 443-446 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
SHORT COMMUNICATION CGA 7214
GAS CHROMATOGRAPHIC BIOLOGICAL FLUIDS
B.L. GOODWIN,
C.R.J. RUTHVEN
ASSAY OF PHENYLACETIC
ACID IN
and M. SANDLER
Bernhard Baron Memorial Research Laboratories and Institute of Obstetrics and Gynaecology, Queen Charlotte’s Maternity Hospital, London W6 OXG (U.K.)
(Received
March 28,1975)
Gas chromatographic (GC) methods are available for the assay of phenylacetic acid (PAA) in urine when the concentration is abnormally high, as in untreated phenylketonurics [l-4] . However, at normal endogenous concentrations these procedures, which use conventional packed columns, are no longer capable of separating PAA from the now proportionately greater amounts of other urinary acids present in normal urine. The use of support-coated open tubular (SCOT) columns appears to offer a solution to these difficulties. However, when TMS ether-esters or alkyl ester-TMS ether derivatives of aromatic acids extracted from urine are chromatographed, PAA, owing to its short retention time, is still incompletely resolved from adjacent peaks. Moreover, retention times are variable and unacceptable tailing occurs because of the prolonged interval at low temperatures required to remove injected solvent before programming to the final running temperature. A critical examination of the factors producing these technical problems has been made which has enabled us to overcome them sufficiently to permit the measurement of PAA at low concentrations in biological fluids. PAA, having only one functional group, can be esterified and readily separated by GC from acids with two or more functional groups; the additional groups on extraneous acids, such as hydroxyl or amino, remain underivatised, resulting in a product with a long retention time. Furthermore, many such acids are not extracted by the technique finally devised. PAA is quite volatile and must be handled as a salt during recovery from solvents by evaporation. However, solvent evaporation may be performed in the presence of the free acid as long as it is mixed with a large excess of non-volatile acids, as in an ether extract from 10 ml urine. The initial ethereal extract (2 X 2.5 vol.) of PAA from an acidified (HCl to pH O-l) biological Correspondence to: Professor M. Sandier. Queen Charlotte’s Maternity Hospital, Goldhawk London W6 OXG, U.K.
Road.
fluid may be evaporated to dryness in the free state under vacuum at 25’C irrespective of the presence of other acids, provided a water pump and not a high vacuum pump is used. Prior to the transfer of PAA and before its esterification, PAA must be converted to a salt. The triethylamine salt was chosen because of the volatility of the amine, the salt being almost non-volatile. One drop of the base is added to the residue from the ether extract and the excess is evaporated. The residue is dissolved in water (2 X 0.15 ml) and each of these portions is transferred successively to a 1 ml Reacti-Vial (Pierce), the second being added after evaporation of the first to dryness in vacua. The final residue is esterified with 0.1 ml alcoholic HCl and left at room temperature for at least 30 min. For routine use, n-propyl esters were found to be better than methyl or ethyl esters, although the last two, with their shorter retention times, might be superior in some situations. Higher esters could not be used because the parent alcohol was insoluble in water (see below). Since esters of PAA are somewhat volatile, the reagent could not be evaporated off either in a stream of gas or in vacua, without incurring significant losses. Direct injection of the reagent was tested following removal of HCl by standing over finely divided anhydrous sodium carbonate (prepared by drying the decahydrate in vacua) but this approach was unsatisfactory as the solvent caused damage to the stationary phase after some days of continuous use. To overcome the difficulty, an extraction procedure was employed after esterifying with n-propanol/HCl, prepared by absorbing HCl gas in n-propanol and standardising to 2.2 N. Propanolic-HCl, prepared by mixing 1.9 ml of acetyl chloride cautiously with 10 ml of propanol, with cooling, could not be employed with urine extract because the propyl acetate formed in its preparation increased the solubility of unwanted esters in n-hexane used in the next stage of the method. n-Propanol/HCl (0.1 ml) was added to the residue containing PAA and after at least 30 min at room temperature, 0.1 ml of n-hexane, containing 0.025% n-hexadecane as an internal standard, was pipetted in, followed by 0.2 ml H,O, and the whole was mixed on a vortex mixer and centrifuged. Pure standards gave two liquid phases, whereas urine extracts often resulted in three liquid phases, an almost colourless upper phase containing the ester of PAA in hexane, a dark phase mainly composed of esters of acids more polar than PAA, and a lower aqueous phase. n-Propyl phenylacetate was stable under these conditions for at least a week. Due to the relative volatility of the ester, and the consequent danger of it emerging from the capillary column mixed with solvent, steps were taken to allow the latter to pass through the column, while the ester remained at the origin, at the same time improving the resolution [ 51. Chromatography was carried out on a 50 ft SCOT column of either SE30 or SE52 when relatively pure samples (e.g. from saliva) were taken, using a pressure of 1.25 psi without a splitter. The column was kept at 70°C for four min after the injection and the temperature was then raised at 32”C/min to 200°C. A volume of 1-2 ~1 was injected. Flame ionization detection was employed. For extracts from unhydrolysed urine, where the presence of other volatile materials interfered, a modified procedure was developed, using 2 X 50 ft SCOT columns linked together. A combination of SE30 and SE52 was found
445
b
MIN
Fig. 1. Chromatograms
of n-propyl
phenylacetate
in (a)
diva
and (b) urine extracts.
to be satisfactory. The initial temperature and rate of heating were kept the same, but the initial time at 70°C was reduced to one min. A gas pressure of 5.5 psi was used. To obtain quantitative results the column was primed before use with 0.1-l pg n-propyl phenylacetate. Compared with the hydrocarbon standard, the derivative is more sensitive to any deterioration of the column and is liable to tail unless conditions remain optimal. It may, therefore, be necessary to inject BSA (say, 2 1.11)from time to time, although excessive treatment can be damaging. When a pressure of about 5.5 psi is used for the 100 ft column, tailing is minimal and a resolution of at least 23 000 plates for hexadecane can be achieved.
Using a 100 ft column, the response was nearly linear relative to the hydrocarbon standard in the normal working range (0.5-20 yg injected), but below this level the response diminished; for an injection of 40 ng, it had decreased by about 40% because of increased tailing at low concentrations. Replicate injections of 1 pg of the acid as its propyl ester gave an acceptable standard deviation of 6.7% for the peak height relative to the hydrocarbon standard. An overall standard deviation of 9.4%, obtained when the acid was extracted from 10 ml of urine containing about 4 pg/ml PAA, compares favourably with the precision of most biochemical assays. The concentration of free acid observed in normal human urine was 0.92 5 0.49 mg/24 h (mean + S.D.) with a range of 0.41-2.02 mg/24 h. After hydrolysis of conjugates (lOO”C, 2 h, 6 N HCl), concentrations of total acid were about a hundred times greater 119 + 31 g/24 h (range 83-165 g/24 h) a value which does not differ substantially from earlier estimates [6,7] . References 1
Blau,
2
Curtius,
3
Wadman, H..
K.
(1970)
Clin.
H.-Ch.,
Vollmin.
S.K.,
Hudson.
4
Karoum,
5
Goodwin.
6
James.
7
Stein,
van
F.P.
F.,
Ruthven,
B.L. M.O.,
W.H.,
and
and
Smith, Paladini,
Chim.
Acta
J.A.
27,
and
5-18
Baerlocher,
K.
Sprang,
F.J.,
van der H&den,
Woolf,
L.I.,
eds)
C.R.J. Sandier. R.L., A.C.,
and
pp.
Sandier,
M. (1975) Williams, Him,
65-72,
R.T. and
Clin.
C. and
Ketting,
Thieme
M. (1968)
Clin.
C.H.
(1972)
and
Chim.
Verlag,
Clin. Acta
Chim.
59,
Reidenberg,
Moore,
Chim.
S. (1954)
Acta
D. (1971)
37,
277.-285
in Phenylketonuria
(Bickel,
Stuttgart Acta
20.
427437
253-254
M. (1972) J. Am.
Proc. Chem.
ROY. Sm.
Sot. 76.
B 182, 2848-2849
25-35
Clinica Chimica Acta, 62 (1975) 443-446 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
SHORT COMMUNICATION CGA 7214
GAS CHROMATOGRAPHIC BIOLOGICAL FLUIDS
B.L. GOODWIN,
C.R.J. RUTHVEN
ASSAY OF PHENYLACETIC
ACID IN
and M. SANDLER
Bernhard Baron Memorial Research Laboratories and Institute of Obstetrics and Gynaecology, Queen Charlotte’s Maternity Hospital, London W6 OXG (U.K.)
(Received
March 28,1975)
Gas chromatographic (GC) methods are available for the assay of phenylacetic acid (PAA) in urine when the concentration is abnormally high, as in untreated phenylketonurics [l-4] . However, at normal endogenous concentrations these procedures, which use conventional packed columns, are no longer capable of separating PAA from the now proportionately greater amounts of other urinary acids present in normal urine. The use of support-coated open tubular (SCOT) columns appears to offer a solution to these difficulties. However, when TMS ether-esters or alkyl ester-TMS ether derivatives of aromatic acids extracted from urine are chromatographed, PAA, owing to its short retention time, is still incompletely resolved from adjacent peaks. Moreover, retention times are variable and unacceptable tailing occurs because of the prolonged interval at low temperatures required to remove injected solvent before programming to the final running temperature. A critical examination of the factors producing these technical problems has been made which has enabled us to overcome them sufficiently to permit the measurement of PAA at low concentrations in biological fluids. PAA, having only one functional group, can be esterified and readily separated by GC from acids with two or more functional groups; the additional groups on extraneous acids, such as hydroxyl or amino, remain underivatised, resulting in a product with a long retention time. Furthermore, many such acids are not extracted by the technique finally devised. PAA is quite volatile and must be handled as a salt during recovery from solvents by evaporation. However, solvent evaporation may be performed in the presence of the free acid as long as it is mixed with a large excess of non-volatile acids, as in an ether extract from 10 ml urine. The initial ethereal extract (2 X 2.5 vol.) of PAA from an acidified (HCl to pH O-l) biological Correspondence to: Professor M. Sandier. Queen Charlotte’s Maternity Hospital, Goldhawk London W6 OXG, U.K.
Road.
fluid may be evaporated to dryness in the free state under vacuum at 25’C irrespective of the presence of other acids, provided a water pump and not a high vacuum pump is used. Prior to the transfer of PAA and before its esterification, PAA must be converted to a salt. The triethylamine salt was chosen because of the volatility of the amine, the salt being almost non-volatile. One drop of the base is added to the residue from the ether extract and the excess is evaporated. The residue is dissolved in water (2 X 0.15 ml) and each of these portions is transferred successively to a 1 ml Reacti-Vial (Pierce), the second being added after evaporation of the first to dryness in vacua. The final residue is esterified with 0.1 ml alcoholic HCl and left at room temperature for at least 30 min. For routine use, n-propyl esters were found to be better than methyl or ethyl esters, although the last two, with their shorter retention times, might be superior in some situations. Higher esters could not be used because the parent alcohol was insoluble in water (see below). Since esters of PAA are somewhat volatile, the reagent could not be evaporated off either in a stream of gas or in vacua, without incurring significant losses. Direct injection of the reagent was tested following removal of HCl by standing over finely divided anhydrous sodium carbonate (prepared by drying the decahydrate in vacua) but this approach was unsatisfactory as the solvent caused damage to the stationary phase after some days of continuous use. To overcome the difficulty, an extraction procedure was employed after esterifying with n-propanol/HCl, prepared by absorbing HCl gas in n-propanol and standardising to 2.2 N. Propanolic-HCl, prepared by mixing 1.9 ml of acetyl chloride cautiously with 10 ml of propanol, with cooling, could not be employed with urine extract because the propyl acetate formed in its preparation increased the solubility of unwanted esters in n-hexane used in the next stage of the method. n-Propanol/HCl (0.1 ml) was added to the residue containing PAA and after at least 30 min at room temperature, 0.1 ml of n-hexane, containing 0.025% n-hexadecane as an internal standard, was pipetted in, followed by 0.2 ml H,O, and the whole was mixed on a vortex mixer and centrifuged. Pure standards gave two liquid phases, whereas urine extracts often resulted in three liquid phases, an almost colourless upper phase containing the ester of PAA in hexane, a dark phase mainly composed of esters of acids more polar than PAA, and a lower aqueous phase. n-Propyl phenylacetate was stable under these conditions for at least a week. Due to the relative volatility of the ester, and the consequent danger of it emerging from the capillary column mixed with solvent, steps were taken to allow the latter to pass through the column, while the ester remained at the origin, at the same time improving the resolution [ 51. Chromatography was carried out on a 50 ft SCOT column of either SE30 or SE52 when relatively pure samples (e.g. from saliva) were taken, using a pressure of 1.25 psi without a splitter. The column was kept at 70°C for four min after the injection and the temperature was then raised at 32”C/min to 200°C. A volume of 1-2 ~1 was injected. Flame ionization detection was employed. For extracts from unhydrolysed urine, where the presence of other volatile materials interfered, a modified procedure was developed, using 2 X 50 ft SCOT columns linked together. A combination of SE30 and SE52 was found
445
b
MIN
Fig. 1. Chromatograms
of n-propyl
phenylacetate
in (a)
diva
and (b) urine extracts.
to be satisfactory. The initial temperature and rate of heating were kept the same, but the initial time at 70°C was reduced to one min. A gas pressure of 5.5 psi was used. To obtain quantitative results the column was primed before use with 0.1-l pg n-propyl phenylacetate. Compared with the hydrocarbon standard, the derivative is more sensitive to any deterioration of the column and is liable to tail unless conditions remain optimal. It may, therefore, be necessary to inject BSA (say, 2 1.11)from time to time, although excessive treatment can be damaging. When a pressure of about 5.5 psi is used for the 100 ft column, tailing is minimal and a resolution of at least 23 000 plates for hexadecane can be achieved.
Using a 100 ft column, the response was nearly linear relative to the hydrocarbon standard in the normal working range (0.5-20 yg injected), but below this level the response diminished; for an injection of 40 ng, it had decreased by about 40% because of increased tailing at low concentrations. Replicate injections of 1 pg of the acid as its propyl ester gave an acceptable standard deviation of 6.7% for the peak height relative to the hydrocarbon standard. An overall standard deviation of 9.4%, obtained when the acid was extracted from 10 ml of urine containing about 4 pg/ml PAA, compares favourably with the precision of most biochemical assays. The concentration of free acid observed in normal human urine was 0.92 5 0.49 mg/24 h (mean + S.D.) with a range of 0.41-2.02 mg/24 h. After hydrolysis of conjugates (lOO”C, 2 h, 6 N HCl), concentrations of total acid were about a hundred times greater 119 + 31 g/24 h (range 83-165 g/24 h) a value which does not differ substantially from earlier estimates [6,7] . References 1
Blau,
2
Curtius,
3
Wadman, H..
K.
(1970)
Clin.
H.-Ch.,
Vollmin.
S.K.,
Hudson.
4
Karoum,
5
Goodwin.
6
James.
7
Stein,
van
F.P.
F.,
Ruthven,
B.L. M.O.,
W.H.,
and
and
Smith, Paladini,
Chim.
Acta
J.A.
27,
and
5-18
Baerlocher,
K.
Sprang,
F.J.,
van der H&den,
Woolf,
L.I.,
eds)
C.R.J. Sandier. R.L., A.C.,
and
pp.
Sandier,
M. (1975) Williams, Him,
65-72,
R.T. and
Clin.
C. and
Ketting,
Thieme
M. (1968)
Clin.
C.H.
(1972)
and
Chim.
Verlag,
Clin. Acta
Chim.
59,
Reidenberg,
Moore,
Chim.
S. (1954)
Acta
D. (1971)
37,
277.-285
in Phenylketonuria
(Bickel,
Stuttgart Acta
20.
427437
253-254
M. (1972) J. Am.
Proc. Chem.
ROY. Sm.
Sot. 76.
B 182, 2848-2849
25-35
