Screening of Organic Acids in Urine by Chemical Ionization Mass Spectrometry David Issachar and Jehuda Yinont Department of Isotope Research, The Weizmann Institute of Science, Rehovot, Israel
A method has been developed for qualitative screening of carboxylic acids in human urine. The method uses direct chemical ionization mass spectrometry with isobutane as reagent gas. The carboxylic acids are separated from the urine samples by ion eychange. Normal carboxylicacid profiles have been determinedand are compared with the profiles of patients suffering from certain metabolic disorders.
mination of amino acids in urine," we have extended this method to provide metabolic profiles for a large number of urinary carboxylic acids.
INTRODUCTION
The investigation of urinary compounds is an essential part of biochemical screening for disorders in the human metabolism. One of the most important groups in urine are the carboxylic acids, which involve and reflect some of the major metabolic functions of the body. Various methods have been proposed for the metabolic screening of the carboxylic acids in human urine to produce a 'metabolic profile'.' Most methods include chromatographic separation of the acidic components followed by mass s e c t r ~ m e t r y l - ~ or ultraviolet absorption techniquess-'for identification. Recently, preliminary experiments have been carried out with various mass spectrometric techniques including field desorption' and chemical ioni~ation.'.'~So far, gas chromatography mass spectrometry is the most frequently used analytical technique for the metabolic screening of body fluids. The disadvantages of GCMS for carboxylic acids are: (1)GCMS is inherently a slow technique, as compared with direct MS. (2) Because of the high polarity of many of the physiologically important carboxylic acids and the low volatility of others, derivatization is often needed, which by itself causes new problems such as increase in the number of compounds," increase in the mass number of the compounds and the possibility of artifacts in the chromatogram." (3) The concentration of some inorganic acids like sulfate and phosphate, normally present in urine, is much higher than the concentration of the organic acids.I3 The inorganic acids are extracted with the organic acids and may lead to ambiguity when TMS derivatives are prepared. The retention indices of TMS derivatives of sulfate and phosphate are in the same range as that of several of the low molecular weight aliphatic carboxylic acids, e.g. lactic, glyceric, 3-hydroxyisovaleric and others. l4 The selective removal of sulfate and phosphate without loss of the organic acids is a very difficult problem. 15316 Recently, several urinary profiles for the acidic components have been published in the literature. 1,4,8-10,15-20 Having demonstrated the use of direct chemical ionization mass spectrometry for the detert Author to whom correspondence should be addressed.
EXPERIMENTAL Patients and methods
Urine samples were obtained from healthy and sick individuals. The age groups studied were: (1) Six infants, 4 weeks old, five of whom were healthy and one suffering from a metabolic disorder. (2)Eight children, 4-10 years old, five of whom were healthy and three suffering from a metabolic disorder. First morning urine specimens were taken and stored at -20 "C until analysed. The longest storage time was five weeks. The acidic metabolites from the urine samples were quantitatively extracted onto a column of Sephadex DEAE-A25 from which they were eluted using a mixture of 0.1 N H C l + E t O H ( 1 : 1). The column of Sephadex DEAE-A25 (7 x 40 mm) was prepared by pouring an aqueous slurry of gel, previously swollen in pyridinium acetate buffer, 0.5 M. An aliquot (2.5 ml) of urine of neutral p H was passed through the column at a flow rate of one drop per 5 s. The column was then washed with 2 x 5 ml of water and the acidic components were eluted with a mixture (11ml) of 0.1 N H C l + E t O H ( 1 : 1). The eluate which contained the acidic components, was concentrated to 0.2-0.5 ml by the freeze drying technique." From this residue an amount of 5 ~1 was introduced through the solid probe, directly into the ion source of the mass spectrometer. The carboxylic acids have been divided into two subgroups, according to their volatility: (1) high volatility carboxylic acids; (2) low volatility carboxylic acids. It is obvious that this division is not sharp, as it is difficult to speak about a specific temperature that can differentiate between the two sub-groups. As a matter of analytical convenience, the high volatility sub-group has been defined as the group including all the carboxylic acids which produce a mass spectrum at a probe temperature
CCC-0306-042X/79/0006-0047$05.00 @ Heyden & Son Ltd, 1979
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
47
D. ISSACHAR AND J. YINON
of less than 60°C (source temperature is higher). The other sub-group contains all the remaining carboxylic acids. The CI mass spectra of the more voltaile carboxylic acids, were taken at a source temperature of 70 "C and a probe temperature of 50 "C. The conditions for recording the CI mass spectra for the less carboxylic acids were as follows: after the peaks corresponding to the more volatile components almost disappeared, the source temperature was elevated to 190°C and probe temperature to 130°C. The mass spectra of the less volatile carboxylic acids was then recorded. The CI mass spectra of the urinary carboxylic acids have been normalized to the peak of creatinine corresponding to 1mg of creatinine. The amount of creatinine present in the 2.5 ml urine sample was determined during the analysis of the amino acids in that sample.21 Quantitation, which was performed every few days, was obtained by integration of the creatinine M + 1 peak at m / z 114 and comparison with a standard sample. Integration was performed by measuring the total area of the peak at m / z 114 from the appearance of the peak until its disappearance. Although the creatinine peak serves as a normalizing peak, it does not appear itself in the mass spectra of the carboxylic acids. This explains the fact that in many spectra there is no peak at 100% relative intensity. Mass spectra were recorded with a Du-Pont 21-490B mass spectrometer equipped with a dual EI/CI source. Isobutane at a pressure of 0.2-0.5 Torr was used as CI reagent gas. Electron emission current was 300 FA and electron energy 300eV. Multiplier gain was lo6 and electron amplifier input resistor lo7 ohm.
xylic acids were from Sigma Chemical Company, USA; Fluka AG, Switzerland and BDH Chemicals Ltd, Poole, UK. Isobutane (high purity grade) was from Liquid Carbonic, Chicago, Illinois, USA. Sephadex DEAEA25 was from Pharmacia, Fine Chemicals AB, Uppsala, Sweden.
RESULTS AND DISCUSSION In order to study the CI mass spectra of the acidic components in urine the CI mass spectrum of each of the urinary components whose concentration in normal urine is higher than g p1-I was recorded. This was done by using various CI reagent gases. As expected, the fragmentation was dependent on the reactivity of the reagent gas and on the ion source temperature. Table 1 presents the CI mass spectra of some physiologically important carboxylic acids with isobutane as reagent gas. By comparing the CI mass spectra obtained with the CI mass spectra of some free carbox$ic acids and their derivative^,^^-^^ and the CI mass spectra of some free amino acids,21,27,28 the characteristic fragmentation of the free carboxylic acids in the CI mode can be summarized as follows (Scheme 1).
6H
I1
R-C-OH+
[R]++ HCOOH
R - C O O H ' V
\
Materials [XH]'
= [C4H9]'.
R +
R-C-OH2+[R--CO]'+H20
[CH5]+or [H301'
All the materials used were analytically pure and obtained from the following companies. All the carbo-
Scheme 1
Table 1. CI isobutane mass spectra of carboxylic acids % Abundance Compound
1 Oxalic 2 Malonic 3 a-Ketoisovaleric 4 a-Hydroxyisovaleric 5 Succinic 6 a-Ketocaproic 7 a-Ketoisocaproic 8 Phenylacetic 9 a-Ketoglutaric 10 Adipic 11 Tartaric 12 Mandelic 13 a-Methoxybenzoic 14 a-Ketoadipic 15 Phenyllactic 16 Vanillic 17 Aconitic 18 Hippuric 19 3.4-Dihydroxymandelic 20 Ascorbic 21 Glucuronic
48
Mol wt
90 104 116 118 118 130 130 136 146 146 150 152 152 160 166 168 174 179 184 176 194
[M+l-H20]+
[M+1-C02]+
[M+l-H2CO,]+
Additional peaks m l z (% abundance)
(25)
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
119(100); 73(20) 91(15) (5) 133(100); 10315) 121(3)
123(5)
113(40); 87(20)
115(5) 159(100); 115(10)
Source temp. ("C)
Probe temp K)
60 60 60 60 60 80 80 80 100 100 200 80 80 150 80 80 200 150 160 220 180
30 30 30 30 30 40 40 40 50 50 140 40
40 100 50 40
130 110 100 140 120
@ Heyden & Son Ltd, 1979
SCREENING OF URINARY ORGANIC ACIDS BY CHEMICAL IONIZATION MASS SPECTROMETRY
For polycarboxylic acids there is a greater loss of formic acid (reaction A) and loss of H 2 0 (reaction B) than in monocarboxylic acids. For carboxyhydroxy acids there is a greater loss of H 2 0 compared with simple carboxylic acids. Carboxyhydroxy acids tend to produce protonated dimers and higher polymer ions at a relatively high intensity. There are several carboxylic acids which produce unusual mass spectra. The fragmentation is usually dependent on the R group present. For example, a ketoisovaleric and a -ketoisocaproic (Table 1)each have a base eak at ( M + 3 ) ( m / z 119 and m / z 133 respectively)29 and not at ( M + 1 ) ( m / z 116 and m / t 130 respectively) as expected.
Figure 3. CI isobutane mass spectrum of mixture c: 1, pyruvic
(rnol. wt 88); 2, phenylacetic (rnol. wt 136); 3, a-ketoglutaric (rnol. wt 146); 4, hydrocinnamic (rnol. wt 150); 5, phenyllactic (mol. wt 166). Source and probe temperatures 70 and 50°C respectively.
Mixture analysis In order to learn about the interactions that may occur amongst the different urinary compounds in the ion source, various standard mixtures have been prepared. The CI mass spectra of some of these mixtures are presented in Fig. 1-4. These CI mass spectra show the existence of mixed protonated dimers, namely the MA + MB+ 1 ions formed in CI (MAand MB are the molecular weights of molecules A and B respectively). The pure protonated dimers (2M+ 1) and mixed protonated dimers also give an indication of the molecular ion peak corresponding to each of the compounds present in the mixture. The criteria for analysis and identification of the peaks in the CI mass spectra of the urine samples were as follows: (1) Recording of the CI mass spectrum of each
m/z Figure 1. CI isobutane mass spectrum of mixture a: 1, n-butyric
(rnol. wt 88); 2, @-OH-butyric(rnol. wt 104); 3, succinic (rnol. wt 118); 4, vanillic (rnol. wt 168); 5, indoleacetic (rnol. wt 175). Source and probe temperatures 70 and 40 "C respectively.
m/r
Figure 4. CI isobutane mass spectrum of mixture d: 1, pyroglutamic (rnol. wt 129); 2, mandelic (rnol. wt 152); 3, p-OH-mandelic (mol. wt 168); 4, p-OH-phenylpyruvic (rnol. wt 180); 5, glucuronic (rnol. wt194); 6, gluconic (mol.wt196). Source and probe temperatures 150 and 100 "C respectively.
o f the pure acids, and comparison of these spectra with the CI spectrum of a urine sample. (2) Accounting for the ions resulting from various ion/molecule interactions occurring in the ion source, when operated in the CI mode. (3) The differences in voltaility for the various components as has been expressed in a crude way by the division into two sub-groups. (4) Temperature dependence of the CI mass spectrum of each of the components. (5) Changes in the CI spectrum when various reagent gases are used. (6) Using the E I mass spectrum of the sample investigated. This is especially efficient for urine components that are in relatively high concentration like hippuric and citric acid. (7) Comparison of the profile of urinary organic acids obtained by CIMS with corresponding profiles recorded by other technique^."^"^-'^ IJrinary carboxylic profiles using CIMS
1 zoo
50
I00 rn/z
Figure2. CI isobutane massspectrum of mixture b: 1, malic (rnol. wt 134); 2, rn-OH-benzoic (mol. wt 138); 3, adipic (rnol. wt 146); 4,
o-MeO-benzoic (rnol. wt 152); 5, 3,4-di-OH-phenylacetic (rnol. wt 168). Source and probe temperatures 90 and 60 "C respectively.
@ Heyden & Son Ltd, 1979
Working under the controlled experimental conditions described previously, CI mass spectra profiles of carboxylic acid which were separated by ion exchange from urine samples were recorded. 'Normal profiles' for the urinary carboxylic acids of healthy infants and children were defined. Table 2 presents the range of peak intensities (and the average) appearing in the CI mass spectra of the urinary organic acids of five healthy infants and five healthy children. The table presents the peaks of both volatile and less volatile carboxylic acids. Some acids produce peaks in both low temperature and high temperature mass spectra. Figure 5 presents the CI mass spectra of the urinary metabolic profiles for the more volatile carboxylic acids BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
49
D. ISSACHAR AND J. YINON ~~~
Table 2. Cl peak intensities of organic acids in normal urinary samples Infants
High temperature
Low temperature
mlz
81 83 84 85 86 87 89 90 91 93 95 97 98 99 101 103 104 105 107 109 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143
Peak intensity
Average
0-40 20-44 0-22 50-1 00
24.6 31.8 7.6 81.4
20-28 0-60 0-8 32-1 20 0-40 0-28 20-56 0-1 6 28-74 32-72 148-280 0-8 24-90 0-1 2 0-1 4 10-24 0-36 44- 100 0-1 2 3690
24.6 20.8 1.6 64.2 14.8 14.2 35.2 6.2 46.8 50.2 209.6 1.6 59.6 8.4 6.4 18.8 18.6 76.0 2.4 51.2
24-54
39.0
240-800 0-48 0-76
442.0 16.8 35.2
0-8
18-52
Children
1.6
33.6
Peak intensity
Average
0-33 0-44 24-50 30-1 00 18-44 12-34 0-22
11.4 21.8 32.8 68.8 32.2 25.8 6.8
0-36 0-8 10-20 38-1 35 0-2 1 24-500 24-36 30-340 0-1 4 10-40 0-1 2 0-1 6 12-22 0-2 1 80-720 0-50 38-1 08 0-1 5 0-22 0-1 6 16-34 0-34
19.6 1.6 13.8 74.2 7.4 149.6 28.6 189.0 2.8 22.2 2.4 7.2 19.8 4.2 271.O 23.4 75.6 5.8 7.6 3.2 23.2 6.8
0-28 12-42 0-32 0-1 8 0-2 1 30-1 00 26-1 00 14-1 50 104-400 40-1 00 0-1 8 0-20
5.6 25.4 11.0 6.6 8.2 61.6 55.4 65.2 228.8 60.6 6.6 10.4
0-24 0-80 18-100
10.4 21.4 52.8
High tenweratwe
Peak intensity
Average
0-20 0-9
9.0 3.8
0-1 2
6.6
0-20 0-30 4-45 4-25
4.4 6.2 25.4 20.6
0-1 6 0-1 0
3.2 3.6
3-1 8 60-1 50 15-66 3-55 0-45
12.2 102.2 47.0
18.6 16.0
Peak intensity
0-20 7-95 10-24
0-5 0-1 8 0-1 40 12-50 7-420 10-1 7 8-59 13-84 0-7 0-7
10 27.6 15.2
1.o 6.0 2.8 25.2 106.4 7 4.4 31.O 42.4 2.2 2.4
22.6
22-1 17
68.0
0-36
17.6
44-85
61.8
20-37
29.4 0-1 8 0-48
7.8 21.8
9.8
24-250 15-32 0-1 0
110.2 25.6 2.0
8-1 20
52.8
6-55 0-1 7 24-60 0-7 30-1 20
29.0 6.6 39.0 1.4 86.0
30-72 0-40 22-54 123-360 28-60
39.0 21.2 36.4 221.6 39.8
0-42
16.0
11-32
18.4
0-75
29.6
15-65
36.6 7.8 88.2 16.6 13.4 16.8
105-320
5-20
204.8
37.2 1.6 101.2 1.6 92.4 2.4 16.2 4.0 16.4
0-20
10.8
30-52
42.0
7-1 6
11.0
16-24
19.2
9.8
22.8
18.2 4.2 17.0
3-1 8
18-28
12-21 0-21 12-21
0-28 38-225 0-38 10-20
7-17
14.0
0-32
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
Average
5-43
28-48 0-8 70-1 60 0-8 64-1 50 0-12 12-20 0-20 12.24
of three of the healthy children and Figs. 6 presents the CI mass spectra of the less volatile carboxylic acids of the same patients. Tables 3 and 4 show the identity of the peaks appearing in Table 2 and in Fig. 5 and 6. Part of the fluctuations 50
Low temperature
in these normal profiles is due to dietary variations as well as to individual metabolic differences. However, these ‘normal profiles’ are constant enough to be distinguished from abnormal profiles of sick patients as will be shown in the next examples. @ Heyden & Son Ltd, 1979
SCREENING OF URINARY ORGANIC ACIDS BY CHEMICAL IONIZATION MASS SPECTROMETRY
Infants
Children
Low temperature
Low temperature
High temperature
high temperature
-
miz
144 145 146 147 149 150 151 153 154 155 157 158 159 160 161 162 163 165 167 168 169 171 172 173 175 177 178 179 180 181 183 185 187 189 190 191 193 195 196 197 199 201 203 208 21 1 215 217 218 224 225 240 247 248 249
Peak intensity
-
Average
36-84 0-4 44-1 12 18-50
56.4 0.8 83.2 31.2
0-48 0-4 0-20 20-50 0-8 36-140 0-8 12-54
23.4 0.8 11.4 32.0 1.6 63.2 1.6 26.4
9-30 0-28
21.4 5.6
0-20 12-20
10.8 14.8
12-26 32-440 0-1 50
18.4 116.8 32.8
0-20
4.0
0-24 0-1 0 0-24 0-1 4
12.6 3.8 11.2 2.8
0-1 8
3.6
0-80 0-30
Peak intensity
Average
0-50 12-24 0-50 12-430 0-44 44-170 0-1 8 8-46
13.0 20.0 13.0 147.8 13.6 82.8 6.0 22.6
22-44 14-130 0-80 22-44
31.0 54.4 30.8 35.2
15-30 0-45 0-1 4
22.6 26.2 2.8
0-40
8.0 20.0 31.1 7.2 14.2 88.0
0.60 0-54 0-36 0-38 12-210
30-200 0-30
18.4 6.0
0-40
101.8 6.0
8.0
12-30 8-30 0-1 0
18.0 16.2
Average
Peak intensity
Average
15-78 0-92 0-1 02 22-198 0-1 08 32-99 0-40 10-60 0-1 2 20-30 3 6 1 35 60-121 22-70
49.2 30.0 20.4 116.6 29.6 54.4 20.0 32.4 2.4 24.2 88.8 83.8 48.0 28.0 270.8 70.8 68.4 13.8
14-1 20
53.8
22-83 15-80
47.6 37.4
0-35 11-45
11.4 35.0
0-1 8 0-34
7.0 15.8
15-65
41 .O
11-50
28.0
7-33 9-70 0-53
19.8 28.2 23.6
0-54 90-725 35-94 18-1 50 0-42
7-85 5-1 5
44.8 10.2
0-1 60 0-42
71.4 8.4
6-47 37-23 1 8-55
24.4 96.2 22.6
35-1 98
136.6
5-60 30-1 65 23-130 12-50 5-50 5-80 9-55
20.8 104.0 62.0 23.8 24.0 34.4 26.0
0-50 0-55 220-990 31-500 0-52 0-42
10.0 14.8 782.0 187.4 17.2 11.2
0-64 0-45
30.2 9.0
0-34 7-58
0-40
11.8 20.2 18.2
0-63
17.2
0-93 0-1 33 0-118 0-55
18.6 43.4 23.8 11.0
0-36 10-70 0-1 15 0-70 0-50 0-3 1
14.8 30.0 51.8 14.0 14.4 10.2
25-1 50 0-1 54
69.4 63.6
0-60 0-200 0-78 0-1 30 64-240 24-160
12.0 40.0 15.6 41 .O 145.6 92.8
0-37
7.4
4.0
Phenylketonuria patient (10 years old)
Figure 7 shows the profiles of the more volatile carboxylic acids of a healthy and a phenylketonuria (PKU) patient. The differences between the two profiles can be seen clearly. In the profile of the PKU patient an increase in the intensity of the following peaks is ob@ Heyden & Son Ltd, 1979
Peak intensity
served: m / z 137 (5-fold), m / z 153 (15-fold), m / z 165 (?'-fold) and m / z 167 (20-fold). The increase in intensity of these peaks is due to the possible increase in excretion of the following acids as expected in a PKU patient:30 phenylacetic, phenylpyruvic ( m / z 137), mandelic, hydroxyphenylacetic ( m / z 153), phenylpyruvic ( m / z 165) and phenyllactic ( m / z 167). BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
51
D. ISSACHAR AND J. YINON
..
60 40
20 -
s
0
100 I
I
180
40
,(;'
, , ,
20
0
~
, I
50
I
* 6.5 781
145
I l l 208
100
150
200
250
I
i
300
m/z
Figure 5(a-c). CI isobutane mass spectra of normal urine samples of children 4-10 years old, showing the metabolic profiles of the volatile carboxylic acids. Source and probe temperatures 70 and 50 "C respectively.
Maple syrup urine disease patient (4 years old) Figure 8 shows the profiles of the more volatile carboxylic acids of a healthy and a maple syrup urine (MSU) disease patient. Differences in the profiles are observed as e ~ p e c t e d . ~In' the MSU patient an increase in the intensity of the following peaks is observed: m / z 91 (15-fold), m / z 103 (7-fold), m / z 105 (7-fold) and
m/z
Figure 6(a-c). CI isobutane mass spectra of normal urine samples of children, 4-10 years old, showing the metabolic profiles of the less volatile carboxylic acids. Source and probe temperatures are 190 and 130 "C respectively.
m / z 133 (6-fold).The increased intensity of these peaks is due to the possible increase in the excretion of the following acids: lactic ( m / e 91), valeric ( m / z 103), hydroxybutyric and/or hydroxyisobutyric ( m j z 105), a - ketoisovaleric, hydroxyvaleric, hydroxyisovaleric
Table 3. Carboxylic acid profile in urine by CI isobutane mass spectrometry Peak ( m l r )
71 75 85
87 89 91
101 103 105 107 113 117
119
123
52
Acid
n-Butyric (ethylacetic) a-Ketoisova Ieri c Propionic (methylacetic) a-Ketoisocaproic Acetopyruvic 0-Hydroxybutyric Pyruvic (a-ketopropionic) n-Buty ric Oxalic Lactic 2-Hydroxyisovaleric Succinic n-Valeric Hydroxybutyric Hydroxyphenylpyruvic a-Keto-3-methylvaleric a-Ketoisovaleric Fumaric a-Ketoisovaleric Caproic Tetronic a-Hydroxyisovaleric a-Ketoisovaleric Benzoic Phenylpyruvic
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
Formula
Mol. wt
88 116 74 130 130 104 88 88 90 90 118 118 102 104 180 130 116 116 116 116 136 118 116 122 164
Origin
[ M + 1 -HZO]' [ M + 1 -H2C02)[M+l]' [ M + 1 -HzCOzI' [ M + 1 -H&O2]* [ M + I -H,O]' [M+l]' [M+11+ [M+l]+ [M+l]' [M+1-CO]' [ M + 1 - HZO]' [M+1]' [M+l]' [M+1-741' ( M + 1 - HZO)' [ M + 1 -31' [M+11' [M+11+ [M+ll' [ M + 1 -H20]'
[M+lI' [M+31' [M+11' [ M + 1 - CHZCO]'
@ Heyden & Son Ltd, 1979
SCREENING OF URINARY ORGANIC ACIDS BY CHEMICAL IONIZATION MASS SPECTROMETRY
Table 3. Carboxylic acid profile in urine by CI isobutane mass spectrometry (continued) Peak ( m l z )
129 131 133
135 137 139 147 149 151 153
155 157 161 163 165 167 169 173 175
179 180 181 183 185 189 203 251 265 277 293 329 331 313
Acid
Adipic a-Ketoglutaric a-Keto-3-methylvaleric Acetopyruvic a-Hydroxyisocaproic a-Ketoisocaproic a-Hydroxy-3-methylvaleric Methoxybenzoic Hydroxyphenylpyruvic Phenylacetic Phenylpyruvic Hydroxybenzoic a-Ketoglutaric Adipic a-Keto-4-methiolbutyric Hydrocinnamic Mandelic Hydroxyphenylacetic Methoxybenzoic Capric Suberic a-Ketoadipic Pimelic Lactic 3-Hydroxy-3-methylglutaric Phenylpyruvic Phenyllactic Dihydroxymandelic Vanillic Capric Suberic Aconitic Gluconic Hippuric Hydroxyphenylpyruvic Lactic Homovanillic Sebacic Aze Iaic Sebacic a-Ketoisovaleric + a-Ketoisocaproic Oxalic or Lactic+Suberic Valeric+Suberic or Aconitic a-Ketoisovaleric Su beric or Acon itic Phenylpyruvic Phenyllactic+ Phenylpyruvic Phenyllactic
+
( m / z 119) and a -ketoisocaproic, a -hydroxyisocaproic and 2-hydroxy-3-methylvalericacid ( m l z 133).
Mot. wf
146 146 130 130 132 130 132 152 180 136 164 138 146 146 148 150 152 152 152 172 174 160 160 90 162 164 166 184 168 172 174 174
Origin
[ M + 1 -H,O]' [ M + 1 -H,O] [M + I ] ' [M+I]' IM + I ] ' [M+ 31' [M+lI+ [ M + 1 - HZO]' [ M + 1 - H2C021' [M+l]' [ M + 1 -CO]' [ M+ I]' [M+l]' [M+l]' [M+l]+ [M+l]+ [M+I]+ [M+I]' [M+Il+ [ M + 1 - H20]+ [ M + 1 - HZO]+ [M+l]' [M+I]+ [2M + 1 - H,O]' [M+I]' [M+l]' [M+l]' [ M + 1 - HZO]' [ M + I]' [M+l]' [M+l]+ [M+lI+
196 179 180 90 182 202 188 202 116,130 90,174 102,174 116,174
[ M + 1 - HZO]' [M+ I ] + [M+I]+ [2M+ 1I' [M+l]+ [ M + 1 - HZO]' [M+ I ] + [M+l]+ [M, + M2+5]' [M, + Mz + I ] ' [M, +M,+lI+ [M, + M, +3]+
164 166,164 166
[2M+11+ [M, + M z + I ] ' [2M + I ] '
droxyisobutyric ( m l z 105), and also hydroxyvaleric, hydroxyisovaleric, a -ketoisovaleric ( m / z 119).
Reye's Syndrome3' patient (4 years old) Unknown disease (infant 4 weeks old) Figure 9 shows the metabolic profiles of the volatile carboxylic acids of a normal (4year old) and a Reye's Syndrome patient. Large differences in the profiles are observed. In the Reye's Syndrome patient's profile an increase in the intensity of the following peaks is observed: m / z 91 (30-fold), m / z 105 (9 fold) and m / z 119 (7-fold). The increase in the intensity of these peaks is due to the possible increase in excretion of the following acids: lactic ( m l z 91), hydroxybutyric and/or hy@ Heyden & Son Ltd, 1979
This example concerns an infant with an unknown disease. From the first day of his life he suffered from severe convulsions. In the hospital, hyperammonemia was observed. Amino acid anal sis by amino acid analyser and by mass spectrometrJ' did not show abnormal concentrations of the amino acids in the blood and urine. An exception was arginine in urine which was 1.5-2 fold the normal amount present in healthy infants at this age. BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
53
D. ISSACHAR AND J. YINON
Table 4. Carboxylic acid profile in urine by CI isobutane mass spectrometry at high temperature mlz
84 85 103 113 129 130 131 144 147 157 159 162 175 180 185 196 203
Compound
Pipecolic Citric Citric Citric Adipic 2-Ketoglutaric Citric Pipecolic Aconitic Citric Aminoadipic Adipic 2-Ketoglutaric Citric Suberic Orotic Citric Glucuronic Hippuric Aminoadipic Suberic Citric Hippuric Sebacic Hydroxyhippuric Sebacic
Mol. wt
129 192 192 192 146 146 192 129 174 192 161 146 146 192 174 156 192 194 179 161 174 192 179 202 195 202
Origin
[ M + 1 - H,COzI+ [ M + 1 -H~COz-COz-Hz01+ [ M 1 - HzC02 - COzl: [M+I-COz-2.HzOI [ M + 1 -HzO]' [ M + I -HzO]' [ M + 1 -HZO-H2C021+ [M+I]+ [M+l-COJ+ [M+I-H20-COzl+ [ M + 1 -Hz01' [M+l]+ [M+11' [ M + 1 - HzCOZ]+ [M+l-HzO]+ [M+I]+ [M+1-2.H,O]: [M+1-2.HzO] [M+l-HZO]' [M+l]+ [M+l]' [ M + 1 -Hz0]' [M+ll+ [ M + 1 - Hz01' [M+1]' [M+lI+
+
We determined the metabolic profile of the urinary carboxylic acids of this infant by the CIMS method just described. Three urine samples taken on three different days were analysed. As a comparison five urine samples from healthy infants (each 4 weeks old) were similarly analysed. Comparison of the corresponding profiles of the healthy and sick infants for the volatile carboxylic acids (Fig. 10)showed no significant differences in the profiles.
2
E
40
6o 40
l
-,
R
0
:i
20 -
m /I
Figure 8. CI isobutane mass spectra of a normal urine sample (a) and a MSU disease patient (b), showing the metabolic profiles of
the volatile carboxylic acids. Peak contribution is due to the following acids: m l z 91, oxalic, lactic and 2-hydroxyisovaleric; m l z 103, n-valeric; rnlz 105, hydroxybutyric; rnlz 119, hydroxyisovaleric, a-ketoisovaleric and tetronic; rnlz 133, hydroxyisocaproic, 2-hydroxy-3-methylvaleric and a-ketoisocaproic. Source and probe temperatures are 70 and 50°C respectively.
However, the less volatile carboxylic acids (Fig. 11) showed differences between the profiles of the healthy and sick infants. The sick infant's profile shows an increased intensity of the following peaks: m / z 85, 103,
60 40
r n
1
4d
I I
L.
I
.
I
, I
50
1
I
-4
I , I !
100
I50
J.
I '
4
200
250
m /I
Figure 7. CI isobutane mass spectra of a normal urine sample (a) and a PKU patient (b), showing the metabolic profiles of the volatile carboxylic acids. Peak contribution is due t o the following acids: rnlz 103, n-valeric; rnlz 119, hydroxyisovaleric, aketoisovaleric and tetronic; m l z 137, phenylacetic and phenylpyruvic; m l z 153, mandelic, hydroxyphenylacetic and methoxybenzoic; m l z 165, phenylpyruvic; m l z 167, phenyllactic and dihydroxymandelic; rnlz 175, suberic and aconitic; rnlz 319, [M, + M z + I]+, mandelic+phenyllactic, hydroxyphenylacetic+ phenyllactic and methoxybenzoic+phenylIactic; rnlz 329 [2M + I]+-phenylpyruvic; rnlz 331, [M, +Mz+I]+, phenylpyruvic+ phenyllactic; rnlz 333, [2M + lit, phenyllactic. Source and probe temperatures 70 and 50°C respectively.
54
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
Figure 9. CI isobutane mass spectra of a normal urine sample (a) and a Reye's Syndrome patient (b), showing the metabolic profiles of the voltaile carboxylic acids. Peak contribution is due t o the following acids: mlz91, oxalic, lactic and 2-hydroxyisovaleric; rnlz 105, hydroxybutyric; rnlz 119, hydroxyisovaleric, a-ketoisovaleric, 2-hydroxy-3-methylvaleric and tetronic; rnlz 133, hydroxyisocaproic and a-ketoisocaproic; m l z 137, phenylacetic and phenylpyruvic; m l z 181, [2M+l]+, oxalic and lactic; m l z 195, [M, + M z + 1]+, oxalic+ hydroxybutyric and lactic+ hydroxybutyric; rnlz 209 [2M + 1]+, hydroxybutyric; rnlz 223, [M, + M z + 1]+, hydroxybutyrict hydroxyisovaleric, hydroxybutyric+a-ketoisovaleric and [M, + M,+ 1 HzO]', hydroxybutyricftetronic; rnlz 237, [2M+ I ] * , hydroxyisovaleric. Source and probe temperatures 70 and 50°C respectively.
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SCREENING OF URINARY ORGANIC ACIDS B Y CHEMICAL IONIZATION MASS SPECTROMETRY
i I m
"
!
I
60
,
1
I
f IZI
40 60[
20 _7
r - T
50
m/z
,,,,
Jl)J 1 ,
-f-
100
150
200
, ,
,\, 250
m/ z
Figure 10. CI isobutane mass spectra of a normal urine sample (a) and an 'unknown sickness' patient (b), showing the metabolic profiles of the volatile carboxylic acids. Source and probe temperatures 70 and 50°C respectively.
113, 129, 131, 147, 157 and 175, which are the characteristic peaks of citric acid. The increase in the concentration of citric acid was confirmed later in the hospital by the acetic anhydride-pyridine method.33 In conclusion, first, the presented method of CIMS of carboxylic acids enables a rapid qualitative determination of some of the acids present in an unknown urine sample, without need of derivatization. This method is superior to G C alone since it is much faster and does not require prior information about the sample as in G C where columns and experimental conditions have to be preset. Second, 'normal profiles' can be determined by CIMS. These profiles can serve as references in the determination of normality or abnormality of the carboxylic acid profile. Information obtained by this method will
Figure 11. CI isobutane mass spectra of a normal urine sample (a) and an 'unknown sickness' patient (b), showing the metabolic profiles of the less voltaile carboxylic acids. Peak contribution is due to the following acids: m l z 8 5 , 103, 113, 129, 131, 147, 157 and 175 are the typical peaks of a CI mass spectrum of citric acid; m/z 130, pipecolic; mlz 147, citric, adipic and 2-ketoglutaric; mlz 157, citric, suberic and orotic; m / z 175, citric and suberic; m l z 2 4 7 and 248 have not been identified. Source and probe temperatures 190 and 130°C respectively.
indicate the next stage of analysis which will usually be a specific analysis of the suspected metabolites. Third, in principle this method can be extended to other body fluids like blood and amniotic fluid.
Acknowledgements 'The authors would like to thank Professor 0. Sperling, Dr F. Reisner and D r J. Amir of the Beilinson Medical Center, Petah Tikva and Dr M. Statter of the Hadassah University Hospital, Jerusalem, for helpful discussions and for supplying the metabolic patient samples.
REFERENCES 1. E. C. Horning and M. G. Horning, Methods Med. Res. 12,368 (1 970). 2. H. H. White, Clin. Chim. Acta 21, 297 (1968). 3. M. Stafford, M. G. Horning and A. Zlatkis, J. Chromatogr. 126, 495 (1976). 4. I. Molner and C. Hovarrth, J. Chromatogr. 143,391 (1977). 5. J. E. Mrocheck, W. C. Butts, W. T. Rainey and C. A. Butris, Clin. Chem. 17,72 (1971). 6. K. Murayama and N. Shindo, J. Chromatogr. (Biomedical Applications) 143, 137 (1977). 7. A. W. Lis, D. J. McLaughlin, R. K. McLaughlin, E.W. Lis and E. G. Stubbs Clin. Chem. 22, 1528 (1976). 8. M. Anbar, R. L. Dyer and M. E. Scolnick. Clin. Chem. 22, 1503 (1976). 9. A. 6. Robinson, M. Weiss, W. E. Reynolds and L. R. Robinson, American Society for Mass Spectrometry, 23rd Annual Conference on Mass Spectrometry and Allied Topics, Houston, Texas (1975) proceedings, p. 182. 10. D. lssachar and J. Yinon, American Society for Mass Spectrometry, 25th Annual Conference on Mass Spectrometry and Allied Topics, Washington, DC (1977). Proceedings, p. 242. 11. E. Jellum, J. Chromatogr. 143,427 (1977).
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12. A. R. Johnson, A. C. Fogerty, R. L. Hood, S. Kozuharov and G. L. Ford, J. Lipid Res. 17, 431 (1976). 13. D. F. Consalazio, R. E. Johnson and L. J. Pecora, in Physiological Measurements of Metabolic Functions in Man, pp. 448-450. McGraw-Hill, New York (1963). 14. J. A. Thompson, Clin. Chem. 23,901 (1977). 15. J. A. Thompson and S. P. Markey, Anal. Chem. 47, 1313 (1975). 16. A. M. Lawson, R. A. Chalmers and R. W. E. Watts Clin. Chem. 22, 1283 (1976). '17. B. A. Knights, M. Legendre, J. L. Laster and J. S. Storer, Clin. Chem. 21,888 (1975). '18. L. Bjorkman, C. McLean and G. Steen, Clin. Chem. 22, 49 (1976). 19. J. A. Thompson, B. S. Miles and P. V. Fennessy, Clin. Chem. 23, 1734 (1977). 20. I. Matsurnoto, T. Shinka, T. Kuhara, S. I. Haraguchi and E. Yamauchi, American Society for Mass Spectrometry, 25th Annual Conference on Mass Spectrometry and Allied Topics, Washington, DC (1977). Proceedings, p. 368. 21. D. lssachar and J. Yinon, Clin. Chim. Acta 73,304 (1976). 22. R. A. Chalmers and R. W. E. Watts, Analyst. 97, 958 (1972). 23. B. Munson and F. H. Field, ./.Am. Chem. SOC.88,4337 (1966).
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
55
D. ISSACHAR AND J. YINON
24. H. M. Fales, G. W. Milne and R. S. Nicholson,Ana/. Chem. 43, 1785 (1971). 25. J. L. Holrnes, Org. Mass Spectrom. 7,341 (1973). 26. R. J. Weinkam and J. Gal, Org. Mass Spectrom. 11, 188 (1976). 27. P. A. Leclercq and D. M. Desiderio, Org. Mass Specrrorn. 7 , 515 (1971). 28. C. W. Tsang and K. G. Harrison, J. Am. Chem. SOC.98,1301 (1976). 29. D. lssachar and J. Yinon, to be published.
31. C. Jakobs, E. Solern, J. Ek. K. Halvorson and E. Jellum, J. Chromatogr. 143,31 (1977). 32. R. D. K. Reye, G. Morgan and J. Baral, Lancer ii,749 (1963). 33. A. F. Spencer and J. M. Lowenstein, Biochem. J. 103, 342 (1967).
30. W. E. Knox, in The Metabolic Basis of Inherited Disease, 3rd Edn ed. by J. B. Stanbury, J. B. Wyngaargen and D. S. Fredrickson, pp. 266-295. McGraw-Hill, New York (1972).
Received I9 June I978
56
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
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A method has been developed for qualitative screening of carboxylic acids in human urine. The method uses direct chemical ionization mass spectrometry with isobutane as reagent gas. The carboxylic acids are separated from the urine samples by ion eychange. Normal carboxylicacid profiles have been determinedand are compared with the profiles of patients suffering from certain metabolic disorders.
mination of amino acids in urine," we have extended this method to provide metabolic profiles for a large number of urinary carboxylic acids.
INTRODUCTION
The investigation of urinary compounds is an essential part of biochemical screening for disorders in the human metabolism. One of the most important groups in urine are the carboxylic acids, which involve and reflect some of the major metabolic functions of the body. Various methods have been proposed for the metabolic screening of the carboxylic acids in human urine to produce a 'metabolic profile'.' Most methods include chromatographic separation of the acidic components followed by mass s e c t r ~ m e t r y l - ~ or ultraviolet absorption techniquess-'for identification. Recently, preliminary experiments have been carried out with various mass spectrometric techniques including field desorption' and chemical ioni~ation.'.'~So far, gas chromatography mass spectrometry is the most frequently used analytical technique for the metabolic screening of body fluids. The disadvantages of GCMS for carboxylic acids are: (1)GCMS is inherently a slow technique, as compared with direct MS. (2) Because of the high polarity of many of the physiologically important carboxylic acids and the low volatility of others, derivatization is often needed, which by itself causes new problems such as increase in the number of compounds," increase in the mass number of the compounds and the possibility of artifacts in the chromatogram." (3) The concentration of some inorganic acids like sulfate and phosphate, normally present in urine, is much higher than the concentration of the organic acids.I3 The inorganic acids are extracted with the organic acids and may lead to ambiguity when TMS derivatives are prepared. The retention indices of TMS derivatives of sulfate and phosphate are in the same range as that of several of the low molecular weight aliphatic carboxylic acids, e.g. lactic, glyceric, 3-hydroxyisovaleric and others. l4 The selective removal of sulfate and phosphate without loss of the organic acids is a very difficult problem. 15316 Recently, several urinary profiles for the acidic components have been published in the literature. 1,4,8-10,15-20 Having demonstrated the use of direct chemical ionization mass spectrometry for the detert Author to whom correspondence should be addressed.
EXPERIMENTAL Patients and methods
Urine samples were obtained from healthy and sick individuals. The age groups studied were: (1) Six infants, 4 weeks old, five of whom were healthy and one suffering from a metabolic disorder. (2)Eight children, 4-10 years old, five of whom were healthy and three suffering from a metabolic disorder. First morning urine specimens were taken and stored at -20 "C until analysed. The longest storage time was five weeks. The acidic metabolites from the urine samples were quantitatively extracted onto a column of Sephadex DEAE-A25 from which they were eluted using a mixture of 0.1 N H C l + E t O H ( 1 : 1). The column of Sephadex DEAE-A25 (7 x 40 mm) was prepared by pouring an aqueous slurry of gel, previously swollen in pyridinium acetate buffer, 0.5 M. An aliquot (2.5 ml) of urine of neutral p H was passed through the column at a flow rate of one drop per 5 s. The column was then washed with 2 x 5 ml of water and the acidic components were eluted with a mixture (11ml) of 0.1 N H C l + E t O H ( 1 : 1). The eluate which contained the acidic components, was concentrated to 0.2-0.5 ml by the freeze drying technique." From this residue an amount of 5 ~1 was introduced through the solid probe, directly into the ion source of the mass spectrometer. The carboxylic acids have been divided into two subgroups, according to their volatility: (1) high volatility carboxylic acids; (2) low volatility carboxylic acids. It is obvious that this division is not sharp, as it is difficult to speak about a specific temperature that can differentiate between the two sub-groups. As a matter of analytical convenience, the high volatility sub-group has been defined as the group including all the carboxylic acids which produce a mass spectrum at a probe temperature
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BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
47
D. ISSACHAR AND J. YINON
of less than 60°C (source temperature is higher). The other sub-group contains all the remaining carboxylic acids. The CI mass spectra of the more voltaile carboxylic acids, were taken at a source temperature of 70 "C and a probe temperature of 50 "C. The conditions for recording the CI mass spectra for the less carboxylic acids were as follows: after the peaks corresponding to the more volatile components almost disappeared, the source temperature was elevated to 190°C and probe temperature to 130°C. The mass spectra of the less volatile carboxylic acids was then recorded. The CI mass spectra of the urinary carboxylic acids have been normalized to the peak of creatinine corresponding to 1mg of creatinine. The amount of creatinine present in the 2.5 ml urine sample was determined during the analysis of the amino acids in that sample.21 Quantitation, which was performed every few days, was obtained by integration of the creatinine M + 1 peak at m / z 114 and comparison with a standard sample. Integration was performed by measuring the total area of the peak at m / z 114 from the appearance of the peak until its disappearance. Although the creatinine peak serves as a normalizing peak, it does not appear itself in the mass spectra of the carboxylic acids. This explains the fact that in many spectra there is no peak at 100% relative intensity. Mass spectra were recorded with a Du-Pont 21-490B mass spectrometer equipped with a dual EI/CI source. Isobutane at a pressure of 0.2-0.5 Torr was used as CI reagent gas. Electron emission current was 300 FA and electron energy 300eV. Multiplier gain was lo6 and electron amplifier input resistor lo7 ohm.
xylic acids were from Sigma Chemical Company, USA; Fluka AG, Switzerland and BDH Chemicals Ltd, Poole, UK. Isobutane (high purity grade) was from Liquid Carbonic, Chicago, Illinois, USA. Sephadex DEAEA25 was from Pharmacia, Fine Chemicals AB, Uppsala, Sweden.
RESULTS AND DISCUSSION In order to study the CI mass spectra of the acidic components in urine the CI mass spectrum of each of the urinary components whose concentration in normal urine is higher than g p1-I was recorded. This was done by using various CI reagent gases. As expected, the fragmentation was dependent on the reactivity of the reagent gas and on the ion source temperature. Table 1 presents the CI mass spectra of some physiologically important carboxylic acids with isobutane as reagent gas. By comparing the CI mass spectra obtained with the CI mass spectra of some free carbox$ic acids and their derivative^,^^-^^ and the CI mass spectra of some free amino acids,21,27,28 the characteristic fragmentation of the free carboxylic acids in the CI mode can be summarized as follows (Scheme 1).
6H
I1
R-C-OH+
[R]++ HCOOH
R - C O O H ' V
\
Materials [XH]'
= [C4H9]'.
R +
R-C-OH2+[R--CO]'+H20
[CH5]+or [H301'
All the materials used were analytically pure and obtained from the following companies. All the carbo-
Scheme 1
Table 1. CI isobutane mass spectra of carboxylic acids % Abundance Compound
1 Oxalic 2 Malonic 3 a-Ketoisovaleric 4 a-Hydroxyisovaleric 5 Succinic 6 a-Ketocaproic 7 a-Ketoisocaproic 8 Phenylacetic 9 a-Ketoglutaric 10 Adipic 11 Tartaric 12 Mandelic 13 a-Methoxybenzoic 14 a-Ketoadipic 15 Phenyllactic 16 Vanillic 17 Aconitic 18 Hippuric 19 3.4-Dihydroxymandelic 20 Ascorbic 21 Glucuronic
48
Mol wt
90 104 116 118 118 130 130 136 146 146 150 152 152 160 166 168 174 179 184 176 194
[M+l-H20]+
[M+1-C02]+
[M+l-H2CO,]+
Additional peaks m l z (% abundance)
(25)
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
119(100); 73(20) 91(15) (5) 133(100); 10315) 121(3)
123(5)
113(40); 87(20)
115(5) 159(100); 115(10)
Source temp. ("C)
Probe temp K)
60 60 60 60 60 80 80 80 100 100 200 80 80 150 80 80 200 150 160 220 180
30 30 30 30 30 40 40 40 50 50 140 40
40 100 50 40
130 110 100 140 120
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SCREENING OF URINARY ORGANIC ACIDS BY CHEMICAL IONIZATION MASS SPECTROMETRY
For polycarboxylic acids there is a greater loss of formic acid (reaction A) and loss of H 2 0 (reaction B) than in monocarboxylic acids. For carboxyhydroxy acids there is a greater loss of H 2 0 compared with simple carboxylic acids. Carboxyhydroxy acids tend to produce protonated dimers and higher polymer ions at a relatively high intensity. There are several carboxylic acids which produce unusual mass spectra. The fragmentation is usually dependent on the R group present. For example, a ketoisovaleric and a -ketoisocaproic (Table 1)each have a base eak at ( M + 3 ) ( m / z 119 and m / z 133 respectively)29 and not at ( M + 1 ) ( m / z 116 and m / t 130 respectively) as expected.
Figure 3. CI isobutane mass spectrum of mixture c: 1, pyruvic
(rnol. wt 88); 2, phenylacetic (rnol. wt 136); 3, a-ketoglutaric (rnol. wt 146); 4, hydrocinnamic (rnol. wt 150); 5, phenyllactic (mol. wt 166). Source and probe temperatures 70 and 50°C respectively.
Mixture analysis In order to learn about the interactions that may occur amongst the different urinary compounds in the ion source, various standard mixtures have been prepared. The CI mass spectra of some of these mixtures are presented in Fig. 1-4. These CI mass spectra show the existence of mixed protonated dimers, namely the MA + MB+ 1 ions formed in CI (MAand MB are the molecular weights of molecules A and B respectively). The pure protonated dimers (2M+ 1) and mixed protonated dimers also give an indication of the molecular ion peak corresponding to each of the compounds present in the mixture. The criteria for analysis and identification of the peaks in the CI mass spectra of the urine samples were as follows: (1) Recording of the CI mass spectrum of each
m/z Figure 1. CI isobutane mass spectrum of mixture a: 1, n-butyric
(rnol. wt 88); 2, @-OH-butyric(rnol. wt 104); 3, succinic (rnol. wt 118); 4, vanillic (rnol. wt 168); 5, indoleacetic (rnol. wt 175). Source and probe temperatures 70 and 40 "C respectively.
m/r
Figure 4. CI isobutane mass spectrum of mixture d: 1, pyroglutamic (rnol. wt 129); 2, mandelic (rnol. wt 152); 3, p-OH-mandelic (mol. wt 168); 4, p-OH-phenylpyruvic (rnol. wt 180); 5, glucuronic (rnol. wt194); 6, gluconic (mol.wt196). Source and probe temperatures 150 and 100 "C respectively.
o f the pure acids, and comparison of these spectra with the CI spectrum of a urine sample. (2) Accounting for the ions resulting from various ion/molecule interactions occurring in the ion source, when operated in the CI mode. (3) The differences in voltaility for the various components as has been expressed in a crude way by the division into two sub-groups. (4) Temperature dependence of the CI mass spectrum of each of the components. (5) Changes in the CI spectrum when various reagent gases are used. (6) Using the E I mass spectrum of the sample investigated. This is especially efficient for urine components that are in relatively high concentration like hippuric and citric acid. (7) Comparison of the profile of urinary organic acids obtained by CIMS with corresponding profiles recorded by other technique^."^"^-'^ IJrinary carboxylic profiles using CIMS
1 zoo
50
I00 rn/z
Figure2. CI isobutane massspectrum of mixture b: 1, malic (rnol. wt 134); 2, rn-OH-benzoic (mol. wt 138); 3, adipic (rnol. wt 146); 4,
o-MeO-benzoic (rnol. wt 152); 5, 3,4-di-OH-phenylacetic (rnol. wt 168). Source and probe temperatures 90 and 60 "C respectively.
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Working under the controlled experimental conditions described previously, CI mass spectra profiles of carboxylic acid which were separated by ion exchange from urine samples were recorded. 'Normal profiles' for the urinary carboxylic acids of healthy infants and children were defined. Table 2 presents the range of peak intensities (and the average) appearing in the CI mass spectra of the urinary organic acids of five healthy infants and five healthy children. The table presents the peaks of both volatile and less volatile carboxylic acids. Some acids produce peaks in both low temperature and high temperature mass spectra. Figure 5 presents the CI mass spectra of the urinary metabolic profiles for the more volatile carboxylic acids BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
49
D. ISSACHAR AND J. YINON ~~~
Table 2. Cl peak intensities of organic acids in normal urinary samples Infants
High temperature
Low temperature
mlz
81 83 84 85 86 87 89 90 91 93 95 97 98 99 101 103 104 105 107 109 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143
Peak intensity
Average
0-40 20-44 0-22 50-1 00
24.6 31.8 7.6 81.4
20-28 0-60 0-8 32-1 20 0-40 0-28 20-56 0-1 6 28-74 32-72 148-280 0-8 24-90 0-1 2 0-1 4 10-24 0-36 44- 100 0-1 2 3690
24.6 20.8 1.6 64.2 14.8 14.2 35.2 6.2 46.8 50.2 209.6 1.6 59.6 8.4 6.4 18.8 18.6 76.0 2.4 51.2
24-54
39.0
240-800 0-48 0-76
442.0 16.8 35.2
0-8
18-52
Children
1.6
33.6
Peak intensity
Average
0-33 0-44 24-50 30-1 00 18-44 12-34 0-22
11.4 21.8 32.8 68.8 32.2 25.8 6.8
0-36 0-8 10-20 38-1 35 0-2 1 24-500 24-36 30-340 0-1 4 10-40 0-1 2 0-1 6 12-22 0-2 1 80-720 0-50 38-1 08 0-1 5 0-22 0-1 6 16-34 0-34
19.6 1.6 13.8 74.2 7.4 149.6 28.6 189.0 2.8 22.2 2.4 7.2 19.8 4.2 271.O 23.4 75.6 5.8 7.6 3.2 23.2 6.8
0-28 12-42 0-32 0-1 8 0-2 1 30-1 00 26-1 00 14-1 50 104-400 40-1 00 0-1 8 0-20
5.6 25.4 11.0 6.6 8.2 61.6 55.4 65.2 228.8 60.6 6.6 10.4
0-24 0-80 18-100
10.4 21.4 52.8
High tenweratwe
Peak intensity
Average
0-20 0-9
9.0 3.8
0-1 2
6.6
0-20 0-30 4-45 4-25
4.4 6.2 25.4 20.6
0-1 6 0-1 0
3.2 3.6
3-1 8 60-1 50 15-66 3-55 0-45
12.2 102.2 47.0
18.6 16.0
Peak intensity
0-20 7-95 10-24
0-5 0-1 8 0-1 40 12-50 7-420 10-1 7 8-59 13-84 0-7 0-7
10 27.6 15.2
1.o 6.0 2.8 25.2 106.4 7 4.4 31.O 42.4 2.2 2.4
22.6
22-1 17
68.0
0-36
17.6
44-85
61.8
20-37
29.4 0-1 8 0-48
7.8 21.8
9.8
24-250 15-32 0-1 0
110.2 25.6 2.0
8-1 20
52.8
6-55 0-1 7 24-60 0-7 30-1 20
29.0 6.6 39.0 1.4 86.0
30-72 0-40 22-54 123-360 28-60
39.0 21.2 36.4 221.6 39.8
0-42
16.0
11-32
18.4
0-75
29.6
15-65
36.6 7.8 88.2 16.6 13.4 16.8
105-320
5-20
204.8
37.2 1.6 101.2 1.6 92.4 2.4 16.2 4.0 16.4
0-20
10.8
30-52
42.0
7-1 6
11.0
16-24
19.2
9.8
22.8
18.2 4.2 17.0
3-1 8
18-28
12-21 0-21 12-21
0-28 38-225 0-38 10-20
7-17
14.0
0-32
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
Average
5-43
28-48 0-8 70-1 60 0-8 64-1 50 0-12 12-20 0-20 12.24
of three of the healthy children and Figs. 6 presents the CI mass spectra of the less volatile carboxylic acids of the same patients. Tables 3 and 4 show the identity of the peaks appearing in Table 2 and in Fig. 5 and 6. Part of the fluctuations 50
Low temperature
in these normal profiles is due to dietary variations as well as to individual metabolic differences. However, these ‘normal profiles’ are constant enough to be distinguished from abnormal profiles of sick patients as will be shown in the next examples. @ Heyden & Son Ltd, 1979
SCREENING OF URINARY ORGANIC ACIDS BY CHEMICAL IONIZATION MASS SPECTROMETRY
Infants
Children
Low temperature
Low temperature
High temperature
high temperature
-
miz
144 145 146 147 149 150 151 153 154 155 157 158 159 160 161 162 163 165 167 168 169 171 172 173 175 177 178 179 180 181 183 185 187 189 190 191 193 195 196 197 199 201 203 208 21 1 215 217 218 224 225 240 247 248 249
Peak intensity
-
Average
36-84 0-4 44-1 12 18-50
56.4 0.8 83.2 31.2
0-48 0-4 0-20 20-50 0-8 36-140 0-8 12-54
23.4 0.8 11.4 32.0 1.6 63.2 1.6 26.4
9-30 0-28
21.4 5.6
0-20 12-20
10.8 14.8
12-26 32-440 0-1 50
18.4 116.8 32.8
0-20
4.0
0-24 0-1 0 0-24 0-1 4
12.6 3.8 11.2 2.8
0-1 8
3.6
0-80 0-30
Peak intensity
Average
0-50 12-24 0-50 12-430 0-44 44-170 0-1 8 8-46
13.0 20.0 13.0 147.8 13.6 82.8 6.0 22.6
22-44 14-130 0-80 22-44
31.0 54.4 30.8 35.2
15-30 0-45 0-1 4
22.6 26.2 2.8
0-40
8.0 20.0 31.1 7.2 14.2 88.0
0.60 0-54 0-36 0-38 12-210
30-200 0-30
18.4 6.0
0-40
101.8 6.0
8.0
12-30 8-30 0-1 0
18.0 16.2
Average
Peak intensity
Average
15-78 0-92 0-1 02 22-198 0-1 08 32-99 0-40 10-60 0-1 2 20-30 3 6 1 35 60-121 22-70
49.2 30.0 20.4 116.6 29.6 54.4 20.0 32.4 2.4 24.2 88.8 83.8 48.0 28.0 270.8 70.8 68.4 13.8
14-1 20
53.8
22-83 15-80
47.6 37.4
0-35 11-45
11.4 35.0
0-1 8 0-34
7.0 15.8
15-65
41 .O
11-50
28.0
7-33 9-70 0-53
19.8 28.2 23.6
0-54 90-725 35-94 18-1 50 0-42
7-85 5-1 5
44.8 10.2
0-1 60 0-42
71.4 8.4
6-47 37-23 1 8-55
24.4 96.2 22.6
35-1 98
136.6
5-60 30-1 65 23-130 12-50 5-50 5-80 9-55
20.8 104.0 62.0 23.8 24.0 34.4 26.0
0-50 0-55 220-990 31-500 0-52 0-42
10.0 14.8 782.0 187.4 17.2 11.2
0-64 0-45
30.2 9.0
0-34 7-58
0-40
11.8 20.2 18.2
0-63
17.2
0-93 0-1 33 0-118 0-55
18.6 43.4 23.8 11.0
0-36 10-70 0-1 15 0-70 0-50 0-3 1
14.8 30.0 51.8 14.0 14.4 10.2
25-1 50 0-1 54
69.4 63.6
0-60 0-200 0-78 0-1 30 64-240 24-160
12.0 40.0 15.6 41 .O 145.6 92.8
0-37
7.4
4.0
Phenylketonuria patient (10 years old)
Figure 7 shows the profiles of the more volatile carboxylic acids of a healthy and a phenylketonuria (PKU) patient. The differences between the two profiles can be seen clearly. In the profile of the PKU patient an increase in the intensity of the following peaks is ob@ Heyden & Son Ltd, 1979
Peak intensity
served: m / z 137 (5-fold), m / z 153 (15-fold), m / z 165 (?'-fold) and m / z 167 (20-fold). The increase in intensity of these peaks is due to the possible increase in excretion of the following acids as expected in a PKU patient:30 phenylacetic, phenylpyruvic ( m / z 137), mandelic, hydroxyphenylacetic ( m / z 153), phenylpyruvic ( m / z 165) and phenyllactic ( m / z 167). BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
51
D. ISSACHAR AND J. YINON
..
60 40
20 -
s
0
100 I
I
180
40
,(;'
, , ,
20
0
~
, I
50
I
* 6.5 781
145
I l l 208
100
150
200
250
I
i
300
m/z
Figure 5(a-c). CI isobutane mass spectra of normal urine samples of children 4-10 years old, showing the metabolic profiles of the volatile carboxylic acids. Source and probe temperatures 70 and 50 "C respectively.
Maple syrup urine disease patient (4 years old) Figure 8 shows the profiles of the more volatile carboxylic acids of a healthy and a maple syrup urine (MSU) disease patient. Differences in the profiles are observed as e ~ p e c t e d . ~In' the MSU patient an increase in the intensity of the following peaks is observed: m / z 91 (15-fold), m / z 103 (7-fold), m / z 105 (7-fold) and
m/z
Figure 6(a-c). CI isobutane mass spectra of normal urine samples of children, 4-10 years old, showing the metabolic profiles of the less volatile carboxylic acids. Source and probe temperatures are 190 and 130 "C respectively.
m / z 133 (6-fold).The increased intensity of these peaks is due to the possible increase in the excretion of the following acids: lactic ( m / e 91), valeric ( m / z 103), hydroxybutyric and/or hydroxyisobutyric ( m j z 105), a - ketoisovaleric, hydroxyvaleric, hydroxyisovaleric
Table 3. Carboxylic acid profile in urine by CI isobutane mass spectrometry Peak ( m l r )
71 75 85
87 89 91
101 103 105 107 113 117
119
123
52
Acid
n-Butyric (ethylacetic) a-Ketoisova Ieri c Propionic (methylacetic) a-Ketoisocaproic Acetopyruvic 0-Hydroxybutyric Pyruvic (a-ketopropionic) n-Buty ric Oxalic Lactic 2-Hydroxyisovaleric Succinic n-Valeric Hydroxybutyric Hydroxyphenylpyruvic a-Keto-3-methylvaleric a-Ketoisovaleric Fumaric a-Ketoisovaleric Caproic Tetronic a-Hydroxyisovaleric a-Ketoisovaleric Benzoic Phenylpyruvic
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
Formula
Mol. wt
88 116 74 130 130 104 88 88 90 90 118 118 102 104 180 130 116 116 116 116 136 118 116 122 164
Origin
[ M + 1 -HZO]' [ M + 1 -H2C02)[M+l]' [ M + 1 -HzCOzI' [ M + 1 -H&O2]* [ M + I -H,O]' [M+l]' [M+11+ [M+l]+ [M+l]' [M+1-CO]' [ M + 1 - HZO]' [M+1]' [M+l]' [M+1-741' ( M + 1 - HZO)' [ M + 1 -31' [M+11' [M+11+ [M+ll' [ M + 1 -H20]'
[M+lI' [M+31' [M+11' [ M + 1 - CHZCO]'
@ Heyden & Son Ltd, 1979
SCREENING OF URINARY ORGANIC ACIDS BY CHEMICAL IONIZATION MASS SPECTROMETRY
Table 3. Carboxylic acid profile in urine by CI isobutane mass spectrometry (continued) Peak ( m l z )
129 131 133
135 137 139 147 149 151 153
155 157 161 163 165 167 169 173 175
179 180 181 183 185 189 203 251 265 277 293 329 331 313
Acid
Adipic a-Ketoglutaric a-Keto-3-methylvaleric Acetopyruvic a-Hydroxyisocaproic a-Ketoisocaproic a-Hydroxy-3-methylvaleric Methoxybenzoic Hydroxyphenylpyruvic Phenylacetic Phenylpyruvic Hydroxybenzoic a-Ketoglutaric Adipic a-Keto-4-methiolbutyric Hydrocinnamic Mandelic Hydroxyphenylacetic Methoxybenzoic Capric Suberic a-Ketoadipic Pimelic Lactic 3-Hydroxy-3-methylglutaric Phenylpyruvic Phenyllactic Dihydroxymandelic Vanillic Capric Suberic Aconitic Gluconic Hippuric Hydroxyphenylpyruvic Lactic Homovanillic Sebacic Aze Iaic Sebacic a-Ketoisovaleric + a-Ketoisocaproic Oxalic or Lactic+Suberic Valeric+Suberic or Aconitic a-Ketoisovaleric Su beric or Acon itic Phenylpyruvic Phenyllactic+ Phenylpyruvic Phenyllactic
+
( m / z 119) and a -ketoisocaproic, a -hydroxyisocaproic and 2-hydroxy-3-methylvalericacid ( m l z 133).
Mot. wf
146 146 130 130 132 130 132 152 180 136 164 138 146 146 148 150 152 152 152 172 174 160 160 90 162 164 166 184 168 172 174 174
Origin
[ M + 1 -H,O]' [ M + 1 -H,O] [M + I ] ' [M+I]' IM + I ] ' [M+ 31' [M+lI+ [ M + 1 - HZO]' [ M + 1 - H2C021' [M+l]' [ M + 1 -CO]' [ M+ I]' [M+l]' [M+l]' [M+l]+ [M+l]+ [M+I]+ [M+I]' [M+Il+ [ M + 1 - H20]+ [ M + 1 - HZO]+ [M+l]' [M+I]+ [2M + 1 - H,O]' [M+I]' [M+l]' [M+l]' [ M + 1 - HZO]' [ M + I]' [M+l]' [M+l]+ [M+lI+
196 179 180 90 182 202 188 202 116,130 90,174 102,174 116,174
[ M + 1 - HZO]' [M+ I ] + [M+I]+ [2M+ 1I' [M+l]+ [ M + 1 - HZO]' [M+ I ] + [M+l]+ [M, + M2+5]' [M, + Mz + I ] ' [M, +M,+lI+ [M, + M, +3]+
164 166,164 166
[2M+11+ [M, + M z + I ] ' [2M + I ] '
droxyisobutyric ( m l z 105), and also hydroxyvaleric, hydroxyisovaleric, a -ketoisovaleric ( m / z 119).
Reye's Syndrome3' patient (4 years old) Unknown disease (infant 4 weeks old) Figure 9 shows the metabolic profiles of the volatile carboxylic acids of a normal (4year old) and a Reye's Syndrome patient. Large differences in the profiles are observed. In the Reye's Syndrome patient's profile an increase in the intensity of the following peaks is observed: m / z 91 (30-fold), m / z 105 (9 fold) and m / z 119 (7-fold). The increase in the intensity of these peaks is due to the possible increase in excretion of the following acids: lactic ( m l z 91), hydroxybutyric and/or hy@ Heyden & Son Ltd, 1979
This example concerns an infant with an unknown disease. From the first day of his life he suffered from severe convulsions. In the hospital, hyperammonemia was observed. Amino acid anal sis by amino acid analyser and by mass spectrometrJ' did not show abnormal concentrations of the amino acids in the blood and urine. An exception was arginine in urine which was 1.5-2 fold the normal amount present in healthy infants at this age. BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
53
D. ISSACHAR AND J. YINON
Table 4. Carboxylic acid profile in urine by CI isobutane mass spectrometry at high temperature mlz
84 85 103 113 129 130 131 144 147 157 159 162 175 180 185 196 203
Compound
Pipecolic Citric Citric Citric Adipic 2-Ketoglutaric Citric Pipecolic Aconitic Citric Aminoadipic Adipic 2-Ketoglutaric Citric Suberic Orotic Citric Glucuronic Hippuric Aminoadipic Suberic Citric Hippuric Sebacic Hydroxyhippuric Sebacic
Mol. wt
129 192 192 192 146 146 192 129 174 192 161 146 146 192 174 156 192 194 179 161 174 192 179 202 195 202
Origin
[ M + 1 - H,COzI+ [ M + 1 -H~COz-COz-Hz01+ [ M 1 - HzC02 - COzl: [M+I-COz-2.HzOI [ M + 1 -HzO]' [ M + I -HzO]' [ M + 1 -HZO-H2C021+ [M+I]+ [M+l-COJ+ [M+I-H20-COzl+ [ M + 1 -Hz01' [M+l]+ [M+11' [ M + 1 - HzCOZ]+ [M+l-HzO]+ [M+I]+ [M+1-2.H,O]: [M+1-2.HzO] [M+l-HZO]' [M+l]+ [M+l]' [ M + 1 -Hz0]' [M+ll+ [ M + 1 - Hz01' [M+1]' [M+lI+
+
We determined the metabolic profile of the urinary carboxylic acids of this infant by the CIMS method just described. Three urine samples taken on three different days were analysed. As a comparison five urine samples from healthy infants (each 4 weeks old) were similarly analysed. Comparison of the corresponding profiles of the healthy and sick infants for the volatile carboxylic acids (Fig. 10)showed no significant differences in the profiles.
2
E
40
6o 40
l
-,
R
0
:i
20 -
m /I
Figure 8. CI isobutane mass spectra of a normal urine sample (a) and a MSU disease patient (b), showing the metabolic profiles of
the volatile carboxylic acids. Peak contribution is due to the following acids: m l z 91, oxalic, lactic and 2-hydroxyisovaleric; m l z 103, n-valeric; rnlz 105, hydroxybutyric; rnlz 119, hydroxyisovaleric, a-ketoisovaleric and tetronic; rnlz 133, hydroxyisocaproic, 2-hydroxy-3-methylvaleric and a-ketoisocaproic. Source and probe temperatures are 70 and 50°C respectively.
However, the less volatile carboxylic acids (Fig. 11) showed differences between the profiles of the healthy and sick infants. The sick infant's profile shows an increased intensity of the following peaks: m / z 85, 103,
60 40
r n
1
4d
I I
L.
I
.
I
, I
50
1
I
-4
I , I !
100
I50
J.
I '
4
200
250
m /I
Figure 7. CI isobutane mass spectra of a normal urine sample (a) and a PKU patient (b), showing the metabolic profiles of the volatile carboxylic acids. Peak contribution is due t o the following acids: rnlz 103, n-valeric; rnlz 119, hydroxyisovaleric, aketoisovaleric and tetronic; m l z 137, phenylacetic and phenylpyruvic; m l z 153, mandelic, hydroxyphenylacetic and methoxybenzoic; m l z 165, phenylpyruvic; m l z 167, phenyllactic and dihydroxymandelic; rnlz 175, suberic and aconitic; rnlz 319, [M, + M z + I]+, mandelic+phenyllactic, hydroxyphenylacetic+ phenyllactic and methoxybenzoic+phenylIactic; rnlz 329 [2M + I]+-phenylpyruvic; rnlz 331, [M, +Mz+I]+, phenylpyruvic+ phenyllactic; rnlz 333, [2M + lit, phenyllactic. Source and probe temperatures 70 and 50°C respectively.
54
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
Figure 9. CI isobutane mass spectra of a normal urine sample (a) and a Reye's Syndrome patient (b), showing the metabolic profiles of the voltaile carboxylic acids. Peak contribution is due t o the following acids: mlz91, oxalic, lactic and 2-hydroxyisovaleric; rnlz 105, hydroxybutyric; rnlz 119, hydroxyisovaleric, a-ketoisovaleric, 2-hydroxy-3-methylvaleric and tetronic; rnlz 133, hydroxyisocaproic and a-ketoisocaproic; m l z 137, phenylacetic and phenylpyruvic; m l z 181, [2M+l]+, oxalic and lactic; m l z 195, [M, + M z + 1]+, oxalic+ hydroxybutyric and lactic+ hydroxybutyric; rnlz 209 [2M + 1]+, hydroxybutyric; rnlz 223, [M, + M z + 1]+, hydroxybutyrict hydroxyisovaleric, hydroxybutyric+a-ketoisovaleric and [M, + M,+ 1 HzO]', hydroxybutyricftetronic; rnlz 237, [2M+ I ] * , hydroxyisovaleric. Source and probe temperatures 70 and 50°C respectively.
@ Heyden & Son Ltd, 1979
SCREENING OF URINARY ORGANIC ACIDS B Y CHEMICAL IONIZATION MASS SPECTROMETRY
i I m
"
!
I
60
,
1
I
f IZI
40 60[
20 _7
r - T
50
m/z
,,,,
Jl)J 1 ,
-f-
100
150
200
, ,
,\, 250
m/ z
Figure 10. CI isobutane mass spectra of a normal urine sample (a) and an 'unknown sickness' patient (b), showing the metabolic profiles of the volatile carboxylic acids. Source and probe temperatures 70 and 50°C respectively.
113, 129, 131, 147, 157 and 175, which are the characteristic peaks of citric acid. The increase in the concentration of citric acid was confirmed later in the hospital by the acetic anhydride-pyridine method.33 In conclusion, first, the presented method of CIMS of carboxylic acids enables a rapid qualitative determination of some of the acids present in an unknown urine sample, without need of derivatization. This method is superior to G C alone since it is much faster and does not require prior information about the sample as in G C where columns and experimental conditions have to be preset. Second, 'normal profiles' can be determined by CIMS. These profiles can serve as references in the determination of normality or abnormality of the carboxylic acid profile. Information obtained by this method will
Figure 11. CI isobutane mass spectra of a normal urine sample (a) and an 'unknown sickness' patient (b), showing the metabolic profiles of the less voltaile carboxylic acids. Peak contribution is due to the following acids: m l z 8 5 , 103, 113, 129, 131, 147, 157 and 175 are the typical peaks of a CI mass spectrum of citric acid; m/z 130, pipecolic; mlz 147, citric, adipic and 2-ketoglutaric; mlz 157, citric, suberic and orotic; m / z 175, citric and suberic; m l z 2 4 7 and 248 have not been identified. Source and probe temperatures 190 and 130°C respectively.
indicate the next stage of analysis which will usually be a specific analysis of the suspected metabolites. Third, in principle this method can be extended to other body fluids like blood and amniotic fluid.
Acknowledgements 'The authors would like to thank Professor 0. Sperling, Dr F. Reisner and D r J. Amir of the Beilinson Medical Center, Petah Tikva and Dr M. Statter of the Hadassah University Hospital, Jerusalem, for helpful discussions and for supplying the metabolic patient samples.
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@ Heyden & Son Ltd, 1979
12. A. R. Johnson, A. C. Fogerty, R. L. Hood, S. Kozuharov and G. L. Ford, J. Lipid Res. 17, 431 (1976). 13. D. F. Consalazio, R. E. Johnson and L. J. Pecora, in Physiological Measurements of Metabolic Functions in Man, pp. 448-450. McGraw-Hill, New York (1963). 14. J. A. Thompson, Clin. Chem. 23,901 (1977). 15. J. A. Thompson and S. P. Markey, Anal. Chem. 47, 1313 (1975). 16. A. M. Lawson, R. A. Chalmers and R. W. E. Watts Clin. Chem. 22, 1283 (1976). '17. B. A. Knights, M. Legendre, J. L. Laster and J. S. Storer, Clin. Chem. 21,888 (1975). '18. L. Bjorkman, C. McLean and G. Steen, Clin. Chem. 22, 49 (1976). 19. J. A. Thompson, B. S. Miles and P. V. Fennessy, Clin. Chem. 23, 1734 (1977). 20. I. Matsurnoto, T. Shinka, T. Kuhara, S. I. Haraguchi and E. Yamauchi, American Society for Mass Spectrometry, 25th Annual Conference on Mass Spectrometry and Allied Topics, Washington, DC (1977). Proceedings, p. 368. 21. D. lssachar and J. Yinon, Clin. Chim. Acta 73,304 (1976). 22. R. A. Chalmers and R. W. E. Watts, Analyst. 97, 958 (1972). 23. B. Munson and F. H. Field, ./.Am. Chem. SOC.88,4337 (1966).
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
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D. ISSACHAR AND J. YINON
24. H. M. Fales, G. W. Milne and R. S. Nicholson,Ana/. Chem. 43, 1785 (1971). 25. J. L. Holrnes, Org. Mass Spectrom. 7,341 (1973). 26. R. J. Weinkam and J. Gal, Org. Mass Spectrom. 11, 188 (1976). 27. P. A. Leclercq and D. M. Desiderio, Org. Mass Specrrorn. 7 , 515 (1971). 28. C. W. Tsang and K. G. Harrison, J. Am. Chem. SOC.98,1301 (1976). 29. D. lssachar and J. Yinon, to be published.
31. C. Jakobs, E. Solern, J. Ek. K. Halvorson and E. Jellum, J. Chromatogr. 143,31 (1977). 32. R. D. K. Reye, G. Morgan and J. Baral, Lancer ii,749 (1963). 33. A. F. Spencer and J. M. Lowenstein, Biochem. J. 103, 342 (1967).
30. W. E. Knox, in The Metabolic Basis of Inherited Disease, 3rd Edn ed. by J. B. Stanbury, J. B. Wyngaargen and D. S. Fredrickson, pp. 266-295. McGraw-Hill, New York (1972).
Received I9 June I978
56
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 2, 1979
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