'Specification The determination of urinary vanylmandelic acid (VMA) ; the major peripheral .metabolite of catecholamines (1) has been "used as a good indicator of the neural and adrenal medullary overactivity especially in the detection and the establishment of the presence of pheochromocytoma in adults (2) and neuroblastoma in children (3) .

Literature abounds with analytical methods for the determination of urinary VMA. These include spectro- photometry (4,5,6), electrophoresis (7,8), paper and thin-layer chromatography (9,10), gas chrom ography (11,12,13) and isotopic dilution (14) - As discussed by Sandier and Ruthven in their review on VMA and homovanillic acid (HVA) methods of analysis (15) , all these techniques suffer one or more of the following disadvantagesi long time for analysis, non-specificity or being sophisticated enough not to warrant their use in most laboratories.

•In spite of these limitations, the spectrophotometr method of Pisano (6) is widely used although ^' it is time-consuming and lends itself to interference by normal metabolites such a p-hydro ymandelic acid, many drugs and gives results that are over 100 per cent higher than results obtained by the more accurate and specific methods such as bidirectional paper chromatography (16) .

Recently, a rapid colorimetric method (17) has been presented for the assay of VMA combining the determination of VMA as an azo dye after Gitlow (18) and the method of Pisano based on the conversion of

VMA to vanillin. Here. ^' again, the values of VMA, although favorably compare with' ^' those, ^" of Pisano, they are still higher than, values reported by the specific methods (16) .

Summar of ^" the Inventio

5 T .-invention constitutes an- improved method for determining urinary vanylmandelic- acid using the Habbal reaction the improvement comprising extracting the diazo derivative of vanylmandelic acid into ethyl acetate, and stabilizing the extract with dimethyIsulfoxide and 2-amino-

^* 3_ 2— ethyl-1-ρropanol to give a stable chromophore which optically absorbs at 600 nm. It is the discovery of this stable chromophore and the formation thereof using the aforementioned reagents which applicant regards as his invention.. Applicant's invention .is not specific 5 concentrations, amounts, etc., and nothing herein shall be read or construed to limit applicant's invention to ^• the specific best mode described.

The present improvement is carried out using conventional spectrophotometrie methods, the invention lying

20 in the discovery that a particular chromophore can be formed stabilized and then determined.using known and ^" established techniques.

Brief Description of the Drawings Figure 1 is the absorption spectrum of the diazo

25 derivative of VMA in various solvents. (1) ethyl acetate, (2) ethyl acetate -t- isopropanol, (3) ethyl acetate ÷ dimethylsulfoxide + 2-amino-2-methyl-l-propanol.

Figure 2 is the calibration curve for the determination of VMS. by the present method.

30 Figure 3 depicts the effect of the diazoniu salt concentration on the absorption spectrum of diazo VMA. (ϋl) - Urine sample ÷ 200 Ul of the diazo reagent, (U2) - The same urine ^' sample + 20 ϋl of the diazo reagent. U1B,U2B are the same as above except that the

35 components of the diazo reagent are added

successively to the reaction mixture.

Figure 4 is the ^' absorption spectrum of urinary VMA measured by our method (urine) and following periodate treatment (blank) . The sample with the internal standard is plotted to confirm the authenticity of urinary VMA.

Figure 5 depicts the correlation between urinary VMA results by the present method and those of Armstrong and Pisano. -The" Be's't Mode

The following procedure is the best mode presently known for carrying out the invention. It must be absolutely understood, however, that the specific quantities, concentrations, material sources, etc., are given solely to set forth the best mode and do not in any measure or any sense limit or define the invention.

%-k"fcs ^' ri'als and Reagents For organic and inorganic compounds and solvents analytical grade reagents are used. VMA, phenol carboxylic acids and other metabolites are purchased from Sigma. Eastman-Kodak (Trademark) Silica Gel thin-layer sheets are used for chromatography. Collection of Urine: A 24 hour sample, free of drugs is used. A simple diet is given. ^' ith no restriction on fruits, beverages or chocolate, preservation is done by the addition of 10 ml of 6N HC1 for each sample before collection.

Reagents A. Potassium carbonate, 111

B. Citrate buffer, 0.2M, pH 4.0

C. P-nitroaniline, 20mM in IN HC1

D. Sodium nitrite, 40mM in water

E. P-nitrobεnzenediazonium chloride, prepared just before use by mixing equal volumes of C and D.

o:-:n

Chrom tographic solvents: I. Benzene, II. Hexane - . -

Benzene-pyridine (65:20:15), III. Benzene - Isopropanol

Acetic acid (60:40:1).

Methods A. Determination of urinary VMA:

1. 2 ml of acidified urine is mixed with 2.0 gm of sodium chloride and extracted with 5.0 ml of ethylacetate for 2 minutes ^' on a Vortex mixer. A second sample of ^* urine with an internal standard of VMA (10 _. UL of 1 mg/ml) is run at the same time.

2. After phase separation, 4.0 ml of ethyl acetate is extracted with 5.0 ml of citrate buffer for 2 minutes. 3. The ethyl acetate layer is discarded and 2.0 ml of the buffer layer is mixed with 3.0 ml of potassium carbonate solution. 20 Ul of. fresh diazoniu reagent is. added and the diazoniu salt of VMA is extracted with 3.0 ml of ethyl acetate after 2 minutes by shaking for 30 seconds ^" . 4 ^" . 2.0 ml of the ethyl acetate is mixed with 1.0 ml of dimethyIsulfoxide and 0.05 ml of. ^■ 2-amino-2— ethyl-1-propanol. 5. The resulting blue color is measured spectrophotometrically at 600 n using water to zero the machine. The sample with the internal standard is used for calculatio Creatinine is done by the Autoanalyzer II technique.

B. Determination of interference by urinary metabolites: 25 ^' Ul of 0.01H of the metabolite is added to 2.0 ml of citric buffer followed by 3.0 ml of- potassium carbonate solution. The following steps are similar to those in A(stens 3,4,5) . '

C. Thin-layer c roπatograph s This is done by the ^' ascending technique. ^' When the solvent front travels to about 1 cm from the- upper edge of the sheet, 'it is air-dried and examined by visual inspection of the colored diazo derivative.

D. Bidirectional paper chromatography: This is done after the method of Armstrong et al. (1) .

E. Spectropho om rie studies and measurements are done.with Varian Spectre-photometer Series 634 equipped with Varian 9176 Model Recorder.

" Results 1. Stability of the diazo derivative of VMA:

When a pure- sample of VMA -is added to citrate buffer and ^" processed as mentioned under Methods, a pink color is extracted with the organic ethyl acetate which fades slowly to a yellow color. As seen in Figur 1, the latter has a maximum at 400 nm of the many attempts used to stabilize the color, isopropyl alcohol offered some protection. However, addition of dimethylsulfoxide and a trace of 2-amino-2-methyl-l-propanol produces a very stable blue chromophore absorbing at a maximum of 600 nm. Of the various reagents tested (KOH, NaOH, H ₃ , diethylamine (DΞA) , triethylamine (TEA) , and 2-amino-2-me hyl-l-propanol, only 2-amino-2-meth l-l- propanol gives the stable blue color with maximum absorbance. Maximum color development is achieved with 1M K-CO-. Ammonium hydroxide (IN) gives a' similar result. The use of IN NaOH destroys the formation of the diazo derivative of V_.1A.

2. Sensitivity and linearity of the method:

As it is seen in Figure 2, the present reaction is linear up to 50 Ug of VMA. A concentration of 5 Ug/ml of citrate buffer gives an absorbance of 0.1

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at ^' 60Q n . The ^" absorbance of the diazo reaction extracted V7ith butanol and measured at 540 h is half that when coiapared with our method.

3. Concentration of the diazonium salt: . Using increasing volumes of the diazo reagent with the same amount of VMA shift the color of the final reaction from blue to green. This is evident in Figure 3 where 10 times concentration of the diazo reagent produces no extra absorbance at 600 nm, but a significant peak at 400 nm. The concentration of the diazo reagent suggested before is enough to react with 15 Ug of VMA/ml of urine-

4. Analytical recoveries: These are shown in Table 1 where various amounts of standards are added to a urine sample. They range from 92% to 110%. (average: 8.6%).

Table 1 Analytical Recovery of Added VMA Added VMA Estimated Concen. Observed Concen. Recover Ug/ g creatininε %

0 - 5.0

5 10 9.2 92

10 15 16.0 106

15 20 18.5 92 20 25 27.5 110

25 30 28.0 93

5. Specificity of the method:

A- Interference studies:

These are done by comparing the intensity of the colors produced by various urinary metabolites with that of VMA at eσui:r.olar concentrations. It is evident from Table 2 that only me anephrin , nor netanephrine, and 3-methoxy 4-hydroxyphenyl glycol produce significant interference.

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^• Tabl ^' 2

^' Urih ^' ayy ^' Me'tabo ^' lite's' Evaluated For Triterfererice

% absorption relati Compound ^{• • '} 'to VMA '(100 ^" ) " " ^'

Vanylmandelic acid 100.0

B-phenyl pyruvic acid 3.3 p-hydroxy phenyl acetic acid 0.0

Ho ovanillic acid 0.0

Gentisic acid 0.0

Pipecolic acid 0

Resorcinol 10.0

P-hydroxymandelic acid 0

Kynurenic acid 0

Xanthurenic acid 0

Pyrocatechol 0

5-Hydroxyindole acetic acid 0

4-hydroxy 3-methoxy phenyl lactic acid 0

Homo gentisic acid 0

Indole 3-acetic acid 0

4-hydroxy 3-methoxy phenyl pyruvic acid .0

4-hydroxy phenyl pyruvic acid 0

Vanillin 0

Normetanephrine 120

Metanephrine 120

HMPG 90

Vanillic acid 26

Anthranillic acid 0 p-hydroxy benzoic acid 0

Kynurenine 0

B-Indole acrylic acid 5.0

3,4 dihtdrox phenyl acetic acid 0

Caffeic acid 0

Ferulic acid 8.0

3-2-lethoxy tyra ine 5.θ ^"

3,4 dihydroxy mandelic acid 5.0

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^• Table 2 '(Cont.) 3- ^' Methαxy tyrosine 0

L-DOPA 0

3-Indole lactic -acid -0

Indole-3 acetic acid 0 B. Assay of urinary VMA following its destruction .by periodate: Since vanillin produces no color (see Table 1) , oxidation of VMA to vanillin by periodate before the addition of the diazo reagent should produce no color in the final analysis-. This is shown in Figure 4 where such treated urine showed no absorbance at.600 nm as in urine blank.

C. Thin-layer chroma ographic studies: These studies are done on the ethyl acetate extract containing the diazo-reactive compound(s) of urine. One spot is seen in all ^' three types of solvents having an _p value of 0.17 in solvent I, 0.27 in solvent II, 0.74 in solvent III, all of which coincide with an authentic sample of

VMA. 6. Statistical analysis of the results:

Statistical analysis of the results obtained by our method in comparison with those from the method of Pisano (6) and of the method of Armstrong et al. (1) is shown in Table 3.

Table 3 Statistical Analysis of Results Obtained 3y The Present Ile hod (Y) And Other Methods (x) Pisano Armstronσ

Regression line Y = 0.480 -f- 0.048 Y = l.lOX-0.305 Coefficient of determination 0.673 0.987

No. of determinations 16 16

A ^Q ---H

Good agreement and correlation is demonstrated between our method and that of Armstrong, but there is a significant difference between our results and those of Pisano. Figure 5 demonstrates the correlation between our method and both procedures.

Discussion The determination of VMA by the diazo method, although quick, is non-specific (18) . The specificity of the present method has resulted from the interplay of two factors: the extraction of ethyl acetate, layer with citrate buffer instead of sodium or potassium carbonate, and the extraction of the diazo derivative of VMA by ethyl acetate instead of butanol as suggested by the non-specific procedure. In fact, I have detected less acids by extraction with citrate buffer than with carbonate solution as has been shown b Gumboldt (17) . However, the greater part of specificity is due to specific stability . -. of diazo VMA by the dimethyIsulfoxide which is supported by thin-laye .chromatographic studies. The interference by metanephrins is negligible since they are not extracted with ethyl acetate from acidified urine and so the case for MHPG which is excreted normally as a water-soluble sulfate ester (19) . The interfering effect of vanillic acid is also eliminated since it is not extracted with citrate buffer from ethyl acetate. Therefore, these specificity studies have proved no interference from endogenous sources. But, one is still faced with interference from an endogenous source which is the diazo reagent itself. It is evident from Figure 3 where increasing the amount of that reagent adds no extra absorbance at 600 nm - but a significant increase at 400 nm. The ^' latter peak is most probably due to p-nitrophanol, a degradation product of the reagent, although it can be argued that being a relatively strong acid, it should not

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be extracted v/ith the ethyl acetate from alkali solutions-. Therefore, in a normal urine with low value of VMA, a high concentration of the diazo reagent will falsely raise that value up to a level comparable with that of Pisano..

The concentration proposed by this method is . enough to react with 15 ϋg of VMA/ l. urine which is - rarely exceeded, and can be increased proportionately when necessary. The high specificity of this present method is reflected by the values obtained when compared with other methods. This is evident from the correlation study done with Armstrong procedure. The latter is well known to correlate with the isotopic dilution method (14).. 5 - It is to be emphasized that the specific best mode disclosed does not in any way or any degree limit or suggest any limitation of the invention to the concentrations, ratios, amounts, etc. of the reagents. Those competent in analytical techniques will recognize 0 that there is enormous variation possible in such variables as overall amount, concentration, etc. For example, it is generally accepted that a component to be determined should be extracted into the smallest amount of extract solvent which will give a good partition of _. 5 h component, provide convenient volumes for handling, and not overly dilute the extracted component, but absolute values, fixed maxima and minima rarely if ever have any place in such techniques. Likewise, it is usually regarded as optimum to provide sufficient excess ^ of a reagent to assure complete reaction of the component to be determined, but not such a great excess as would interfere with quantitative measurements. In many instances, very large excess' are used to give a constant background measurement for reagent contribution to the 5 measurement. Thus, the precise values in amounts, volumes, concentrations, ratios, etc. are simply the best mode

^C: - ^:?I

arrived at by the inventor .and do not represent, in any sense the inventive concept and nothing herein- shall be ^' so construed. Without attempting any definite limitations, and based upon predictions, from experience with the reagents and chromophore involved and competence in the technical field in which this invention lies, it is projected that the ratio of ethyl acetate extract"of the diazo derivative of vanylmandelic acid to other stabilizing reagents will generally be in the range of from about 0.1 to about 0.9 parts of dimethylsulfoxide and 0-01 to 0.5 part, and' usually from 0.01 to 0.1 part, of 2-amino-2-me hyl-l-propanol per part of such extract, all by volume. But, as discussed, these are general ranges and not critical to the invention nor do these ranges constitute the invention, the discovery or any essential part thereof.

Industrial Application This invention finds practical and industrial application in h spitals and clinics generally throughout the world where diagnostic tests are performed.

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' Re'fe'rences

1. Armstrong, M.D. , McMillan, A. , and. K,N.F. Shaw (1957) . Biochim. Biophys . Acta-. 25, 422-423.

2. Swerdloff , R.3. (1972) . Cal. Med. 117, 44-49 ^' .

3. Von Studnitz, W. , Kaser, H-, Sjoerds ^' a, A. (1963 ⁾ New Eng. J. Med. ^" 269, 232-235.

4. Sandier, M., Ruthven, C.R.J. (1959). Lancet ii, 1034.

5. Sandier, M., Ruthven, C.R.J. (1961). Biochem. J. 80, 78-82.

6. Pisano, J.J., Crout, J.R., Abraham, D- - (1962) . Clin- 0 Chim. Acta 7, 285-291.

7- Klein, D., Chernaik, J.M. (1961). Clin. Che . 7,

257-264. 8. Eichhorn, F., Rutehberg, A. (1963). Clin- Chem. 9, ^' 615-619. 5 9. Armstrong, M.D., Shaw, K.N.F., Wall, P.E. (1956). J. Biol. Chem. 218, 293-303. 10. ^• Annino, J.S., Lipson, M., Williams, L-A. (1965),. Clin. Chem. 11, 905-913,

11 Sprinkle, T.J., Porter, A.H., Gr-et, M..William, C-, ^' -

20 (1969) . ^' Clin. Chim. Acta 25, 409-411.

12 Kahane, Z., Mowat, J.H., Vestergaard, ^* . P. (1969). Clin- Chi . Acta 26, 307-311.

13 Angaard, E., Sedrall, G. (1969). Anal. Chem. 41, 1250-1256.

25 14 Weise, V., K. , Mcdonald, R-K-, Labrosse, Ξ.H. (1961). Clin. Chim. Acta 6, 79-86.

15. Sandier, M., Ruthven, C.R.J. (1966). Ph ^' ar . Rev. 18, 343-351.

16. Gittow, S.E., Mendlo itz, M., Wilk, F..K., Wilk, S.,

->.?. Wolf, R.L., Bertani, L-M. (1963). J. Lab. Clin. lied. 72 612-620. •

17. • GumboIdt,. G. (1977). CΪin. Chem. 23, 1949-1950. 13. Gitlow, E.S., Bertani, L.,M., Ra sen A., (1970).

Cancer, 25, 1377-1383.

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19. Bigelow, L.B., ^' Neal, S, Weil-Malherbe, M. (1971) J. Lab. Clin. ∑ted. 77, .677-683...

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