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[8] Determination of methylmalonic acid in biological fluids by mass spectrometry
[8]
GC-MS
ASSAY OF METHYMALONIC ACID
47
[8] D e t e r m i n a t i o n o f M e t h y l m a l o n i c A c i d in B i o l o g i c a l Fluids by Mass Spectrometry B y JANE A . M O N T G O M E R Y
and ORVALA.
MAMER
Introduction Methylmalonyl-CoA is a metabolite produced during the catabolism of valine and isoleucine by the carboxylation of propionyl-CoA by propionyl-CoA carboxylase (EC 6.4.1.3). Under normal circumstances, small amounts of methylmalonyl-CoA are hydrolyzed and free methylmalonic acid (MMA) can be measured in urine and in serum) Greatly increased concentrations of MMA may occur when inheritance or other causes lead to faulty or insufficient synthesis of any of several enzymes and factors involved with the metabolism of methylmalonyl-CoA. Some of these are methylmalonyl-CoA mutase (EC 5.4.99.2) which converts R-methylmalonyl-CoA to succinyl-CoA, enzymes responsible for conversion of dietary vitamin B12 to adenosylcobalamin required by the mutase, and intrinsic factor and other proteins required for cobalamin absorption and transport. Gas chromatography-mass spectrometry (GC-MS) procedures for MMA have been reported previously. A stable isotope assay has been developed using 2-[methyl-2H3]methylmalonicacid (MMAD3) as internal standard, methyl ester derivatization, and selected ion monitoring detection (SIM) under chemical ionization conditions.2 A second employs ethylmalonic acid as internal standard, cyclohexyl ester derivatives, electron impact ionization, and SIM. 3 A third assay has been reported using MMADa, the trimethylsilyl (TMS) esters, electron impact ionization, and SIM determination of the ratio of ions characteristic of the ratio MMA: MMAD3. 4,5 The first two methods have a variety of difficulties and inherent sources of error that the third method was developed to avoid. TMS derivatives have the advantage over methyl esters in that they are more easily and safely prepared and provide TMS ethers of hydroxylated and enolizable keto acids that are more stable and amenable to GC conditions of analysis. Furthermore, if the MMA concentration is found to be 1 j. A. Montgomery, O. A. Mamer, and C. R. Seriver, J. Clin. Invest. 72, 1937 (1983). 2 A. B. Zinn, D. G. Hine, M. J. Mahoney, and K. Tanaka, Pediatr. Res. 16, 740 (1982). 3 E. J. Norman, H. K. Berry, and M. D. Denton, Biomed. Mass Spectrom. 6, 546 (1979). 4 F. K. Trefz, H. Schmidt, B. Tauscher, E. Depene, R. Baumgartner, G. Hammersen, and W. Kochen, Fur. J. Pediatr. 137, 261 (1981). 5 j. A. Montgomery, M.Sc. thesis. McGill University, Montreal, Quebec, Canada, 1982.
METHODS IN ENZYMOI-DGY,VOL. 166
Col~ight © 1988by AcademicPress,Inc. Allrightsof~'productionin any form reserved.
48
ANALYTICAL AND SYNTHETIC METHODS
[8]
normal, the remaining silylate may be used for conventional organic acid profiling for other organic acids. The high temperatures and acidic conditions required for the preparation of the cyclohexyl esters are severe compared to those for silylation, and risk decarboxylative loss of MMA. MMAD 3 is better than ethylmalonic acid (EMA) as internal standard because it is chemically much more similar to MMA, and it avoids the problem of the occurrence of endogenous EMA in concentrations greater than normal concentrations for MMA in both urine and serum. Electron impact ionization is used; it is nearly universally available in mass spectrometry laboratories and avoids the problems of reestablishing chemical ionization conditions that reproduce the same ratio of M +" to [M + H] + required to avoid uncertainties in the measurement of MMAD3 due to natural heavy isotope abundance incorporation in endogenous MMA. Assay Principle This is a stable isotope dilution assay which measures MMA against a known amount of MMAD3 as the internal standard. The peak representing the coeluting internal standard and endogenous MMA is examined with GC inlet under SIM conditions. The most intense fragments that display the isotopic labeling are selected, as these intensities affect the ultimate sensitivity of the assay. The relative proportions of the labeled and unlabeled ions are determined and are related to concentrations using a calibration curve. Preparation of M M A D a Internal Standard A standard malonate ester synthesis is employed. Sodium ethoxide is produced by dissolving 230 rag of sodium metal (10 mmol) in 20 ml of absolute ethanol in a 50-ml round-bottom flask. When the sodium is completely dissolved, 500 mg of diethyl malonate (3.1 mmol) is added to the flask with stirring. [2H3]Methyliodide (500 mg, 3.4 mmol) is taken up in a syringe and added dropwise with stirring to the flask. A reflux condenser is attached and the reaction is heated at reflux for 5 hr. The mixture is transferred to a 100-ml beaker containing l0 ml of water and the ethanol is allowed to evaporate in a gentle airstream overnight. Rotary evaporation of the ethanol is not recommended as the diethyl methylmalonate is volatile. The ester is saponified by adding 20 ml of water and 1 g of sodium hydroxide (25 mmol) and stirring at room temperature for 12 hr. Residual ester is removed by extracting the basic saponificate three times with 20 ml of diethyl ether (extract is discarded). The product mixture will be contaminated with the unreaeted malonic acid and the dialkylated product,
[8]
G C - M S ASSAY OF METHYMALONIC ACID
49
2,2-[dimethyl-2H6]dimethylmalofflc acid (DMMAD6). The latter is differentially extracted from the acidified aqueous phase (pH 2 or less with HC1) with three 10-ml volumes of diethyl ether/hexane (1:3). This extract should be retained until it has been ascertained that the MMAD3 has been satisfactorily recovered. MMAD3 is isolated by extracting the acidified reaction mixture three times with diethyl ether alone (20 ml). The extract is dried over anhydrous MgSO4 and the ether is removed by rotary evaporation. The crude MMAD 3 is recrystallized from hot benzene. The yield should be at least 50%. Residual DMMAD 6 and malonic acid contamination can be determined by GC-MS by making and analyzing the TMS esters of approximately 20/~g of the product under the conditions of the assay described below. The malonic acid peak will elute well resolved from MMAD3 and DMMAD6, which coelute, and the ratio of total ion current may be used to relate the ratio of malonic to the other two acids. The ratio of MMAD3 to DMMAD6 may in turn be estimated from the ratio of their respective [M-CH3] + peaks, m/z 250 and 267, which bear similar fractions of the total ion current for each derivative. The degree of purity should be in excess of 80%, and this factor is to be taken into account when weighing this material. Calibration Curve A known solution of the internal standard is prepared by dissolving 40 mg of MMAD3 in 10 ml ethyl acetate. Since each analytical sample is prepared using 20 gg internal standard, this stock solution will be sufficient for approximately 2000 analyses. Two stock solutions of unlabeled MMA having concentrations 0.01 #g//tl and 1.0 #g/gl (I and II, respectively) are prepared by dissolution of l0 mg of MMA in l0 ml of distilled water, and subsequent dilution of 0.10 ml of this solution to 10 ml. The eight mixtures described in Table I should produce a calibration curve suitable for fluids such as urine. The acids are recovered by acidifying the solutions with 10% HC1 and extracting them three times with 1-ml volumes of diethyl ether. The ether extracts are dried over MgSO4 and evaporated under a gentle stream of nitrogen. The TMS derivatives are made and these are analyzed by GC-MS as described below. The blank sample allows the determination of the amount of unlabeled MMA in the internal standard. If the blank is comparable to the quantity of endogenous MMA present in the sample (i.e., > 2%) it will adversely affect the lower limit of reliable quantitation. The blank is typically of the order of 1%, and must be determined each time a new batch of MMAD3 is synthesized.
50
ANALYTICAL AND SYNTHETIC METHODS
[8]
TABLE I CALIBRATION CURVE FOR METHYLMALONIC ACIDa Unlabeled MMA Sample No.
Stock Solution
Vol (/tl)
Weight (/zg)
1 2 3 4 5 6 7 8
I I I II II II II Blank
20 50 100 5 10 20 50 0
0.2 0.5 1.0 5.0 10.0 20.0 50.0 0
a All samples contain 5/zl of the internal standard solution (20/zg of MMAD3) and varying amounts of the unlabeled stock solutions I and II (concentrations 0.01 and 1.0/~g//tl). All samples are diluted to 1 ml with distilled water.
Figure 1 shows a typical calibration curve prepared by the above method. The logarithms ofthe observed ratios of the signals of the m/z 247 to 250 ions (ordinate) are plotted against the logarithms of the known weights of unlabeled MMA added for each of the eight mixtures prepared. The lower end of the curve is nonlinear due to the unlabeled content of the internal standard and the ordinate approaches the limiting blank value asymptotically. The upper end of the curve is linear (slope = 1.0211, yintercept = - 1.3513, r -- 0.9989). Sample P r e p a r a t i o n
General Extraction Procedure For fluids having low protein concentrations, such as urine, that are expected to have near normal concentrations of MMA, 2-ml aliquots produce ratios of endogenous MMA to MMAD3 suitable for this analysis. If inherited methylmalonic aciduria is suspected, a smaller aliquot (0.5 ml or less) must be used to avoid overwhelming the MMAD3 internal standard. A constant quantity of internal standard (20/tg) is added to each of the samples contained in 5- to I 0-ml centrifuge tubes that may be closed tightly with Teflon-lined screw caps. If necessary, these are diluted to 2 ml with distilled water and shaken to ensure sample homogeneity. The sample
[8]
GC-MS ASSAY OF METHYMALONIC ACID
51
°/
y
Log R~.~ 25O
-1
/"
/"
1..//" -2
-I
Log vg MMA
l
Fro. l. Sample calibration curve for M M A analysis. The logarithm of observed ratio of the intensifies or areas for m/z 247: 250 (uncombed for isotopic impurities) is plotted versus the logarithm of the known amount of unlabeled MMA added to each calibration mixture (see Table I). For subsequent samples, one locates the point on the calibration curve having as ordinate the logarithm of the observed m/z 247: 250 area ratio. This point will have as the
abscissa the logarithm of the weight of MMA (in micrograms)contained in the sample. Plotting of the ratiosand weightson log-log graph paper may be preferredas an alternative. is saturated with approximately 1 g of sodium chloride and made basic (pH 12- 14) by adding five drops of 10% aqueous sodium hydroxide. It must be verified that the pH is greater than 12. The sample is extracted twice with 2 ml of diethyl ether with vortex mixing to remove basic and neutral components that cause interference at low concentrations. A small inexpensive benchtop centrifuge may be used to separate the organic and aqueous phases of the resulting emulsion. The ether layer is discarded. The sample is acidified with l0 drops of 10% HC1 and checked to ensure that the pH is less than 2. The sample is extracted three times with 2 ml diethyl ether as above. The ether layers are combined, dried over approximately 500 mg of anhydrous MgSO4, and concentrated to 0.5 ml by evaporation under a stream of dry nitrogen. The extract is transferred to a 0.5-ml vial that may be sealed with a Teflon-lined cap and reduced to dryness under a slow nitrogen stream. In the case of protein-rich fluids (CSF, serum, amniotic fluid, culture and perfusing media), it is important to avoid the formation of intractable emulsions by precipitating the protein before starting. As many fluids other than urine contain lower concentrations of MMA, larger aliquots may be required. To each aliquot is added 20 gg of MMAD3 and these are shaken to mix thoroughly. The protein is precipitated by adding 5 - 1 0 drops of saturated aqueous sulfosalicylic acid, mixing well, and then centrifuging to
52
ANALYTICAL AND SYNTHETIC METHODS
[8]
form a proteinaceous pellet. The clear supernatant is transferred to a clean centrifuge tube and the extraction is continued as described for urine samples. Derivatization To the extract residue is added 80 gl of TRISIL/BSA (Pierce Chemical Co., Rockford, I1.), a commercial trimethylsilylating reagent containing N,O-bis(trimethylsilyl)acetamide. The resulting mixtures are heated at 60 ° for 15 min. Analysis
Equipment Requirements A GC-MS instrument is required that is capable of SIM in electron impact mode and data presentation either in an ion current plot integrated by a data system or in an oscillographic recording that allows measurement of the relative intensities of the ions of interest. Analytical conditions are described below.
Gas Chromatography Separation of methylmalonic acid from interfering endogenous organic acids can be accomplished using a 2 m × 2 m m i.d. glass column packed with 6% OV-17 on Chromosorb W/HP. Dimethylsilicone (e.g., SE-30, OV-1, and OV-101) is unsuitable because the TMS derivative of MMA coelutes on this liquid phase with those for 3-hydroxyisovaleric and 2methyl-3-hydroxybutyric acids, both of which have fragment ions common to MMA. The carrier gas is helium and flow rates are optimized for best separator operation (20 ml/min approximately). MMA has a retention time of about 5 min at an isothermal column temperature of 110 °. Column conditioning is important, and this is easily done by making several injections of a sample under analytical conditions before beginning an analysis.
Mass Spectrometry The mass spectra of the bis-TMS derivatives of unlabeled MMA and MMAD3 are reproduced in Fig. 2. Moderately intense ions characteristic of the two compounds are the [M-CH3] + ions at m/z 247 and 250 for endogenous and labeled MMA, respectively. These are the ions selected for the quantitation described here. The ions at m/z 218 and 221 may also be
[8]
G C - M S ASSAY OF METHYMALONIC ACID
53
10014~
73
--
A
o o It H II TMSOC-C-COTMS
CH~
218 . . t
0
I
100.
'"
.~1
L
.
247
L
147
73
B
TMSOC-C-COTMS C~H3
50
22~
250
I
0
100
m/z
200
300
FIG. 2. The 70 eV electron-impact mass spectra of the TMS derivatives of MMA and MMAD3 by GC inlet under the conditions of the assay. The ions selected for quantitative purposes are m/z 247 and 250, and are the fragments formed by the loss of a CH3 radical from one of the TMS groups in the molecular ions of MMA and MMAD3, respectively. The labeled methyl group appears to be refractory to this loss. The molecular ions (m/z 262 and 265, respectively) are present but have intensifies too low to be useful. Subsidiary ions for monitoring are m/z 218 and 221, formed by the loss of CO2 from the molecular ions This last ion pair is used for confirmation of peak identity only, and not for quantitative purposes because column bleed and siloxanes often present a fragment at m/z 221 of highly variable intensity in their spectra.
monitored to confirm the identity of the peak as MMA. A number of peaks will be seen to occur along the m/z 247 trace, but only one will correspond in time to large responses at m/z 218, 221, and 250. Where small amounts of MMA are expected, it is advisable to monitor only m/z 247 and 250, representative of endogenous MMA and MMAD3. This will allow increased sampling time at m/z 247 and therefore more accurate area measurement. The sampling frequency and dwell time per ion should be
54
ANALYTICAL AND SYNTHETIC METHODS
[8]
chosen so that at least 10 samples are taken in the time interval between the half-height points of the peak of interest. This frequency is approximately 3 Hz with the column and conditions described above. Dwell times per ion should be chosen to permit measurements of their intensities at this rate. Suggested are 0.100 sec for both m/z 247 and 250, and 0.050 sec each for m/z 218 and 221. Quantitation Measurement of peak area is more reliable than peak height because of the signal averaging effect that SIM produces. Determinations made using areas will be more accurate than those made with peak heights at low concentrations where signals are small. The logarithm of the ratio of the observed areas for m/z 247:250 is determined and the point on the calibration curve having this value as ordinate will have as abscissa the logarithm of the weight in micrograms of MMA in the extracted volume of the sample. This can be converted to standard concentration values by the following equations: MMA (raM) MMA (mg/g creatinine) =
Weight MMA (#g) Volume extracted (ml) × I 18
(1)
Weight MMA (#g) X 100 Volume extracted (ml) × creatinine (rag%)
(2) It is most useful to use the linear portion of the calibration curve. If very low ratios of MMA: MMADa are obtained consistently (for example when analyzing CSF or perfusate media), potential errors arising from the use of the lower nonlinear part of the curve may be avoided by increasing the volume of sample extracted, or by establishing another calibration curve using a smaller quantity of MMAD3. Using 4 pg of internal standard, for example, instead of 20 gg will increase the m/z 247: 250 intensity ratios fivefold and also reduce the blank MMAD3 contribution to m/z 247. In the case of very low concentrations of MMA, the lower limit of addition of MMAD3 will depend on losses on the gas chromatographic column, which are usually greater for packed columns than for capillary. It will also depend of course on the sensitivity of the mass spectrometer. Conclusion Using this method, the concentration of MMA in random samples of urine of normal adults was found to be 3.27 mg/g creatinine (standard deviation, 4.19 mg/g creatinine; n --- 23).
[9]
ANALYSIS OF A C Y L - C O A ESTERS
55
While newborn screening programs for MMA are well established, there is increased interest in MMA excretion in adult populations where vitamin Bt2 deficiency or malabsorption is suspected to contribute to chronic neurological disorders. This method provides a rapid, specific, and sensitive assay for monitoring or screening patients with these disorders and may also be used to assay MMA in a wide variety of other fluids. A simple modification of this assay will also allow the measurement of ethylmalonic acid (EMA), which is a metabolite of2S,3R-isoleucine [L( + )alloisoleucine]6 and also the hydrolysis product of the carboxylation of butyryl-CoA by propionyl-CoA carboxylase. 7 The internal standard used is 2-[elhyl-2Hs]ethylmalonic acid (EMADs) which can be synthesized in a manner analogous to that for MMAD3, by substituting [2Hs]ethyl iodide for [2H3]methyl iodide. The analysis is performed in SIM using ions m/z 261 and 266 for EMA and EMADs, respectively, and calibrations and measurements analogous to those used for MMA. As most mass spectrometers will allow the monitoring of more than one group of ions, EMA and MMA can be determined simultaneously in one sample simply by adding both internal standards prior to extraction. 6 0 . A. Mamer, S. S. Tjoa, C. R. Scriver, and G. A. Klassen, Biochem. J. 160, 417 (1976). 7 C. S. Hegre, D. R. Halenz, and M. D. Lane, J. Am. Chem. Soc. 81, 6526 (1959).
[9] A n a l y s i s o f A c y l - C o e n z y m e A E s t e r s in B i o l o g i c a l Samples B y BARBARA E. CORKEY
Estimates of acylcoenzyme A (CoAp content of tissues have generally been restricted to the enzymatic analysis of CoASH and acetyl-CoA in the acid-soluble fraction of tissue extracts and of total CoASH, after alkaline hydrolysis of the esters, in the acid-soluble and insoluble fractions of tissue extracts. 2-5 Before the development of high-performance liquid chromatographic (HPLC) methods, specific assays had been developed for only a See "also M. T. King, P. D. Reiss, and N. W. CorneU, this volume [10]; K. Bortlett and A. G. Causey, this volume [11]. 2 j. R. Williamson and B. E. Corkey, this series, Vol. 13, p. 434. 3 p. K. Tubbs and P. B. Garland, this series, Vol. 13, p. 535. 4 j. R. WiUiamson and B. E. Corkey, this series, Vol. 55, p. 200. s A. Olbrich, B. Diet, and F. Lynen, Anal. Biochem. 113, 386 (1981).
METHODS IN ENZYMOLOGY, VOL. 166
Copyright© 1988by AcademicPress,Inc. All rightsof reproduction in any form reserved.
GC-MS
ASSAY OF METHYMALONIC ACID
47
[8] D e t e r m i n a t i o n o f M e t h y l m a l o n i c A c i d in B i o l o g i c a l Fluids by Mass Spectrometry B y JANE A . M O N T G O M E R Y
and ORVALA.
MAMER
Introduction Methylmalonyl-CoA is a metabolite produced during the catabolism of valine and isoleucine by the carboxylation of propionyl-CoA by propionyl-CoA carboxylase (EC 6.4.1.3). Under normal circumstances, small amounts of methylmalonyl-CoA are hydrolyzed and free methylmalonic acid (MMA) can be measured in urine and in serum) Greatly increased concentrations of MMA may occur when inheritance or other causes lead to faulty or insufficient synthesis of any of several enzymes and factors involved with the metabolism of methylmalonyl-CoA. Some of these are methylmalonyl-CoA mutase (EC 5.4.99.2) which converts R-methylmalonyl-CoA to succinyl-CoA, enzymes responsible for conversion of dietary vitamin B12 to adenosylcobalamin required by the mutase, and intrinsic factor and other proteins required for cobalamin absorption and transport. Gas chromatography-mass spectrometry (GC-MS) procedures for MMA have been reported previously. A stable isotope assay has been developed using 2-[methyl-2H3]methylmalonicacid (MMAD3) as internal standard, methyl ester derivatization, and selected ion monitoring detection (SIM) under chemical ionization conditions.2 A second employs ethylmalonic acid as internal standard, cyclohexyl ester derivatives, electron impact ionization, and SIM. 3 A third assay has been reported using MMADa, the trimethylsilyl (TMS) esters, electron impact ionization, and SIM determination of the ratio of ions characteristic of the ratio MMA: MMAD3. 4,5 The first two methods have a variety of difficulties and inherent sources of error that the third method was developed to avoid. TMS derivatives have the advantage over methyl esters in that they are more easily and safely prepared and provide TMS ethers of hydroxylated and enolizable keto acids that are more stable and amenable to GC conditions of analysis. Furthermore, if the MMA concentration is found to be 1 j. A. Montgomery, O. A. Mamer, and C. R. Seriver, J. Clin. Invest. 72, 1937 (1983). 2 A. B. Zinn, D. G. Hine, M. J. Mahoney, and K. Tanaka, Pediatr. Res. 16, 740 (1982). 3 E. J. Norman, H. K. Berry, and M. D. Denton, Biomed. Mass Spectrom. 6, 546 (1979). 4 F. K. Trefz, H. Schmidt, B. Tauscher, E. Depene, R. Baumgartner, G. Hammersen, and W. Kochen, Fur. J. Pediatr. 137, 261 (1981). 5 j. A. Montgomery, M.Sc. thesis. McGill University, Montreal, Quebec, Canada, 1982.
METHODS IN ENZYMOI-DGY,VOL. 166
Col~ight © 1988by AcademicPress,Inc. Allrightsof~'productionin any form reserved.
48
ANALYTICAL AND SYNTHETIC METHODS
[8]
normal, the remaining silylate may be used for conventional organic acid profiling for other organic acids. The high temperatures and acidic conditions required for the preparation of the cyclohexyl esters are severe compared to those for silylation, and risk decarboxylative loss of MMA. MMAD 3 is better than ethylmalonic acid (EMA) as internal standard because it is chemically much more similar to MMA, and it avoids the problem of the occurrence of endogenous EMA in concentrations greater than normal concentrations for MMA in both urine and serum. Electron impact ionization is used; it is nearly universally available in mass spectrometry laboratories and avoids the problems of reestablishing chemical ionization conditions that reproduce the same ratio of M +" to [M + H] + required to avoid uncertainties in the measurement of MMAD3 due to natural heavy isotope abundance incorporation in endogenous MMA. Assay Principle This is a stable isotope dilution assay which measures MMA against a known amount of MMAD3 as the internal standard. The peak representing the coeluting internal standard and endogenous MMA is examined with GC inlet under SIM conditions. The most intense fragments that display the isotopic labeling are selected, as these intensities affect the ultimate sensitivity of the assay. The relative proportions of the labeled and unlabeled ions are determined and are related to concentrations using a calibration curve. Preparation of M M A D a Internal Standard A standard malonate ester synthesis is employed. Sodium ethoxide is produced by dissolving 230 rag of sodium metal (10 mmol) in 20 ml of absolute ethanol in a 50-ml round-bottom flask. When the sodium is completely dissolved, 500 mg of diethyl malonate (3.1 mmol) is added to the flask with stirring. [2H3]Methyliodide (500 mg, 3.4 mmol) is taken up in a syringe and added dropwise with stirring to the flask. A reflux condenser is attached and the reaction is heated at reflux for 5 hr. The mixture is transferred to a 100-ml beaker containing l0 ml of water and the ethanol is allowed to evaporate in a gentle airstream overnight. Rotary evaporation of the ethanol is not recommended as the diethyl methylmalonate is volatile. The ester is saponified by adding 20 ml of water and 1 g of sodium hydroxide (25 mmol) and stirring at room temperature for 12 hr. Residual ester is removed by extracting the basic saponificate three times with 20 ml of diethyl ether (extract is discarded). The product mixture will be contaminated with the unreaeted malonic acid and the dialkylated product,
[8]
G C - M S ASSAY OF METHYMALONIC ACID
49
2,2-[dimethyl-2H6]dimethylmalofflc acid (DMMAD6). The latter is differentially extracted from the acidified aqueous phase (pH 2 or less with HC1) with three 10-ml volumes of diethyl ether/hexane (1:3). This extract should be retained until it has been ascertained that the MMAD3 has been satisfactorily recovered. MMAD3 is isolated by extracting the acidified reaction mixture three times with diethyl ether alone (20 ml). The extract is dried over anhydrous MgSO4 and the ether is removed by rotary evaporation. The crude MMAD 3 is recrystallized from hot benzene. The yield should be at least 50%. Residual DMMAD 6 and malonic acid contamination can be determined by GC-MS by making and analyzing the TMS esters of approximately 20/~g of the product under the conditions of the assay described below. The malonic acid peak will elute well resolved from MMAD3 and DMMAD6, which coelute, and the ratio of total ion current may be used to relate the ratio of malonic to the other two acids. The ratio of MMAD3 to DMMAD6 may in turn be estimated from the ratio of their respective [M-CH3] + peaks, m/z 250 and 267, which bear similar fractions of the total ion current for each derivative. The degree of purity should be in excess of 80%, and this factor is to be taken into account when weighing this material. Calibration Curve A known solution of the internal standard is prepared by dissolving 40 mg of MMAD3 in 10 ml ethyl acetate. Since each analytical sample is prepared using 20 gg internal standard, this stock solution will be sufficient for approximately 2000 analyses. Two stock solutions of unlabeled MMA having concentrations 0.01 #g//tl and 1.0 #g/gl (I and II, respectively) are prepared by dissolution of l0 mg of MMA in l0 ml of distilled water, and subsequent dilution of 0.10 ml of this solution to 10 ml. The eight mixtures described in Table I should produce a calibration curve suitable for fluids such as urine. The acids are recovered by acidifying the solutions with 10% HC1 and extracting them three times with 1-ml volumes of diethyl ether. The ether extracts are dried over MgSO4 and evaporated under a gentle stream of nitrogen. The TMS derivatives are made and these are analyzed by GC-MS as described below. The blank sample allows the determination of the amount of unlabeled MMA in the internal standard. If the blank is comparable to the quantity of endogenous MMA present in the sample (i.e., > 2%) it will adversely affect the lower limit of reliable quantitation. The blank is typically of the order of 1%, and must be determined each time a new batch of MMAD3 is synthesized.
50
ANALYTICAL AND SYNTHETIC METHODS
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TABLE I CALIBRATION CURVE FOR METHYLMALONIC ACIDa Unlabeled MMA Sample No.
Stock Solution
Vol (/tl)
Weight (/zg)
1 2 3 4 5 6 7 8
I I I II II II II Blank
20 50 100 5 10 20 50 0
0.2 0.5 1.0 5.0 10.0 20.0 50.0 0
a All samples contain 5/zl of the internal standard solution (20/zg of MMAD3) and varying amounts of the unlabeled stock solutions I and II (concentrations 0.01 and 1.0/~g//tl). All samples are diluted to 1 ml with distilled water.
Figure 1 shows a typical calibration curve prepared by the above method. The logarithms ofthe observed ratios of the signals of the m/z 247 to 250 ions (ordinate) are plotted against the logarithms of the known weights of unlabeled MMA added for each of the eight mixtures prepared. The lower end of the curve is nonlinear due to the unlabeled content of the internal standard and the ordinate approaches the limiting blank value asymptotically. The upper end of the curve is linear (slope = 1.0211, yintercept = - 1.3513, r -- 0.9989). Sample P r e p a r a t i o n
General Extraction Procedure For fluids having low protein concentrations, such as urine, that are expected to have near normal concentrations of MMA, 2-ml aliquots produce ratios of endogenous MMA to MMAD3 suitable for this analysis. If inherited methylmalonic aciduria is suspected, a smaller aliquot (0.5 ml or less) must be used to avoid overwhelming the MMAD3 internal standard. A constant quantity of internal standard (20/tg) is added to each of the samples contained in 5- to I 0-ml centrifuge tubes that may be closed tightly with Teflon-lined screw caps. If necessary, these are diluted to 2 ml with distilled water and shaken to ensure sample homogeneity. The sample
[8]
GC-MS ASSAY OF METHYMALONIC ACID
51
°/
y
Log R~.~ 25O
-1
/"
/"
1..//" -2
-I
Log vg MMA
l
Fro. l. Sample calibration curve for M M A analysis. The logarithm of observed ratio of the intensifies or areas for m/z 247: 250 (uncombed for isotopic impurities) is plotted versus the logarithm of the known amount of unlabeled MMA added to each calibration mixture (see Table I). For subsequent samples, one locates the point on the calibration curve having as ordinate the logarithm of the observed m/z 247: 250 area ratio. This point will have as the
abscissa the logarithm of the weight of MMA (in micrograms)contained in the sample. Plotting of the ratiosand weightson log-log graph paper may be preferredas an alternative. is saturated with approximately 1 g of sodium chloride and made basic (pH 12- 14) by adding five drops of 10% aqueous sodium hydroxide. It must be verified that the pH is greater than 12. The sample is extracted twice with 2 ml of diethyl ether with vortex mixing to remove basic and neutral components that cause interference at low concentrations. A small inexpensive benchtop centrifuge may be used to separate the organic and aqueous phases of the resulting emulsion. The ether layer is discarded. The sample is acidified with l0 drops of 10% HC1 and checked to ensure that the pH is less than 2. The sample is extracted three times with 2 ml diethyl ether as above. The ether layers are combined, dried over approximately 500 mg of anhydrous MgSO4, and concentrated to 0.5 ml by evaporation under a stream of dry nitrogen. The extract is transferred to a 0.5-ml vial that may be sealed with a Teflon-lined cap and reduced to dryness under a slow nitrogen stream. In the case of protein-rich fluids (CSF, serum, amniotic fluid, culture and perfusing media), it is important to avoid the formation of intractable emulsions by precipitating the protein before starting. As many fluids other than urine contain lower concentrations of MMA, larger aliquots may be required. To each aliquot is added 20 gg of MMAD3 and these are shaken to mix thoroughly. The protein is precipitated by adding 5 - 1 0 drops of saturated aqueous sulfosalicylic acid, mixing well, and then centrifuging to
52
ANALYTICAL AND SYNTHETIC METHODS
[8]
form a proteinaceous pellet. The clear supernatant is transferred to a clean centrifuge tube and the extraction is continued as described for urine samples. Derivatization To the extract residue is added 80 gl of TRISIL/BSA (Pierce Chemical Co., Rockford, I1.), a commercial trimethylsilylating reagent containing N,O-bis(trimethylsilyl)acetamide. The resulting mixtures are heated at 60 ° for 15 min. Analysis
Equipment Requirements A GC-MS instrument is required that is capable of SIM in electron impact mode and data presentation either in an ion current plot integrated by a data system or in an oscillographic recording that allows measurement of the relative intensities of the ions of interest. Analytical conditions are described below.
Gas Chromatography Separation of methylmalonic acid from interfering endogenous organic acids can be accomplished using a 2 m × 2 m m i.d. glass column packed with 6% OV-17 on Chromosorb W/HP. Dimethylsilicone (e.g., SE-30, OV-1, and OV-101) is unsuitable because the TMS derivative of MMA coelutes on this liquid phase with those for 3-hydroxyisovaleric and 2methyl-3-hydroxybutyric acids, both of which have fragment ions common to MMA. The carrier gas is helium and flow rates are optimized for best separator operation (20 ml/min approximately). MMA has a retention time of about 5 min at an isothermal column temperature of 110 °. Column conditioning is important, and this is easily done by making several injections of a sample under analytical conditions before beginning an analysis.
Mass Spectrometry The mass spectra of the bis-TMS derivatives of unlabeled MMA and MMAD3 are reproduced in Fig. 2. Moderately intense ions characteristic of the two compounds are the [M-CH3] + ions at m/z 247 and 250 for endogenous and labeled MMA, respectively. These are the ions selected for the quantitation described here. The ions at m/z 218 and 221 may also be
[8]
G C - M S ASSAY OF METHYMALONIC ACID
53
10014~
73
--
A
o o It H II TMSOC-C-COTMS
CH~
218 . . t
0
I
100.
'"
.~1
L
.
247
L
147
73
B
TMSOC-C-COTMS C~H3
50
22~
250
I
0
100
m/z
200
300
FIG. 2. The 70 eV electron-impact mass spectra of the TMS derivatives of MMA and MMAD3 by GC inlet under the conditions of the assay. The ions selected for quantitative purposes are m/z 247 and 250, and are the fragments formed by the loss of a CH3 radical from one of the TMS groups in the molecular ions of MMA and MMAD3, respectively. The labeled methyl group appears to be refractory to this loss. The molecular ions (m/z 262 and 265, respectively) are present but have intensifies too low to be useful. Subsidiary ions for monitoring are m/z 218 and 221, formed by the loss of CO2 from the molecular ions This last ion pair is used for confirmation of peak identity only, and not for quantitative purposes because column bleed and siloxanes often present a fragment at m/z 221 of highly variable intensity in their spectra.
monitored to confirm the identity of the peak as MMA. A number of peaks will be seen to occur along the m/z 247 trace, but only one will correspond in time to large responses at m/z 218, 221, and 250. Where small amounts of MMA are expected, it is advisable to monitor only m/z 247 and 250, representative of endogenous MMA and MMAD3. This will allow increased sampling time at m/z 247 and therefore more accurate area measurement. The sampling frequency and dwell time per ion should be
54
ANALYTICAL AND SYNTHETIC METHODS
[8]
chosen so that at least 10 samples are taken in the time interval between the half-height points of the peak of interest. This frequency is approximately 3 Hz with the column and conditions described above. Dwell times per ion should be chosen to permit measurements of their intensities at this rate. Suggested are 0.100 sec for both m/z 247 and 250, and 0.050 sec each for m/z 218 and 221. Quantitation Measurement of peak area is more reliable than peak height because of the signal averaging effect that SIM produces. Determinations made using areas will be more accurate than those made with peak heights at low concentrations where signals are small. The logarithm of the ratio of the observed areas for m/z 247:250 is determined and the point on the calibration curve having this value as ordinate will have as abscissa the logarithm of the weight in micrograms of MMA in the extracted volume of the sample. This can be converted to standard concentration values by the following equations: MMA (raM) MMA (mg/g creatinine) =
Weight MMA (#g) Volume extracted (ml) × I 18
(1)
Weight MMA (#g) X 100 Volume extracted (ml) × creatinine (rag%)
(2) It is most useful to use the linear portion of the calibration curve. If very low ratios of MMA: MMADa are obtained consistently (for example when analyzing CSF or perfusate media), potential errors arising from the use of the lower nonlinear part of the curve may be avoided by increasing the volume of sample extracted, or by establishing another calibration curve using a smaller quantity of MMAD3. Using 4 pg of internal standard, for example, instead of 20 gg will increase the m/z 247: 250 intensity ratios fivefold and also reduce the blank MMAD3 contribution to m/z 247. In the case of very low concentrations of MMA, the lower limit of addition of MMAD3 will depend on losses on the gas chromatographic column, which are usually greater for packed columns than for capillary. It will also depend of course on the sensitivity of the mass spectrometer. Conclusion Using this method, the concentration of MMA in random samples of urine of normal adults was found to be 3.27 mg/g creatinine (standard deviation, 4.19 mg/g creatinine; n --- 23).
[9]
ANALYSIS OF A C Y L - C O A ESTERS
55
While newborn screening programs for MMA are well established, there is increased interest in MMA excretion in adult populations where vitamin Bt2 deficiency or malabsorption is suspected to contribute to chronic neurological disorders. This method provides a rapid, specific, and sensitive assay for monitoring or screening patients with these disorders and may also be used to assay MMA in a wide variety of other fluids. A simple modification of this assay will also allow the measurement of ethylmalonic acid (EMA), which is a metabolite of2S,3R-isoleucine [L( + )alloisoleucine]6 and also the hydrolysis product of the carboxylation of butyryl-CoA by propionyl-CoA carboxylase. 7 The internal standard used is 2-[elhyl-2Hs]ethylmalonic acid (EMADs) which can be synthesized in a manner analogous to that for MMAD3, by substituting [2Hs]ethyl iodide for [2H3]methyl iodide. The analysis is performed in SIM using ions m/z 261 and 266 for EMA and EMADs, respectively, and calibrations and measurements analogous to those used for MMA. As most mass spectrometers will allow the monitoring of more than one group of ions, EMA and MMA can be determined simultaneously in one sample simply by adding both internal standards prior to extraction. 6 0 . A. Mamer, S. S. Tjoa, C. R. Scriver, and G. A. Klassen, Biochem. J. 160, 417 (1976). 7 C. S. Hegre, D. R. Halenz, and M. D. Lane, J. Am. Chem. Soc. 81, 6526 (1959).
[9] A n a l y s i s o f A c y l - C o e n z y m e A E s t e r s in B i o l o g i c a l Samples B y BARBARA E. CORKEY
Estimates of acylcoenzyme A (CoAp content of tissues have generally been restricted to the enzymatic analysis of CoASH and acetyl-CoA in the acid-soluble fraction of tissue extracts and of total CoASH, after alkaline hydrolysis of the esters, in the acid-soluble and insoluble fractions of tissue extracts. 2-5 Before the development of high-performance liquid chromatographic (HPLC) methods, specific assays had been developed for only a See "also M. T. King, P. D. Reiss, and N. W. CorneU, this volume [10]; K. Bortlett and A. G. Causey, this volume [11]. 2 j. R. Williamson and B. E. Corkey, this series, Vol. 13, p. 434. 3 p. K. Tubbs and P. B. Garland, this series, Vol. 13, p. 535. 4 j. R. WiUiamson and B. E. Corkey, this series, Vol. 55, p. 200. s A. Olbrich, B. Diet, and F. Lynen, Anal. Biochem. 113, 386 (1981).
METHODS IN ENZYMOLOGY, VOL. 166
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