J. W. Jenne, E. Wyze, F. S. Rood, and F. M. MacDonald, Clin. Pbarmacol. Tber., 13, 349 (1972). J. A. Schack and S. H. Waxler, J. Pharmacol. fxp. Ther., 97, 283 (1949). R. C. Gupta and G. D. Lundberg, Anal. Chem., 45, 2403 (1973). V. P. Shah and S. Riegelman. J. Pharm. Sci., 63, 1283 (1974). R. Osiewicz. V. Aggarwal, R. M. Young and I. Sunshine, J. Chromatogr. Sci., 88, 157 (1974). G. F. Johnson, W. A. Dechtiaruk, and H. M. Soioman. Clin. Chem., 21, 144 (1975). R. D. Thompson and H. T. Nagasawa, J. Lab. Ciin. Med.. 84, 584 (1974). C. V. Manion, D. W. Shoeman. and D. L. Azarnoff, J. Cbromafogr., 101, 169 (1974). M. Weinberger and C. Chidsey, Ciin. Chem., 21/7, 834 (1975). "The United States Pharmacopeia", 19th Rev., Mack Publishing Co.. Easton, Pa., 1975, p 654. P. A . Mitenko and R. I. Ogiivie. Clin. Pharmacol. Ther., 13, 329 (1972).
sensitivity for direct analysis. With sufficiently sensitive detectors, the concentrating function of extraction should not be necessary. Second, a properly designed extraction procedure prevents introduction into the analytical system of many substances that might interfere with the analysis. As a matter of philosophy, however, a high efficiency chromatographic column should be capable of serving in this capacity. Last, extraction precludes introduction into the system those materials (e.g., plasma proteins) that might subsequently interfere with the efficiency of the analytical system. A properly selected molecular filtration system serves effectively to prevent this occurrence. I t would appear, therefore, t h a t such a system as described here would be of use in the non-extractive analysis of a number of drugs.
RECEIVEDfor review August 22, 1975. Accepted October
LITERATURE CITED (1)
24, 1975.
E. S. Vessel1 and G. T. Passananti, Clin. Chem., 17, 851 (1971).
Application of Inexpensive Equipment for High Pressure Liquid Chromatography to Assays for Taurine, y-Amino Butyric Acid, and 5Hydroxytryptophan James L. Meek Laboratory of Preclinical Pharmacology, National lnstitute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032
An inexpensive pump is described for high pressure llquid chromatography which can be assembled from readily available parts. This pressurized tank type apparatus can be used with conventional liquid chromatography detectors, or with the spectrophotometers or fluorometers available in most laboratories. The system has been applied to measurement of specific amino acids in brain tissue: taurine, yaminobutyric acid and 5-hydroxytryptophan. The assays, which can measure less than 5 0 pmol, require only 3-7 min per sample, and require no sample preparation, other than precipitation of proteins. The apparatus can perform complex separations for analytical work, but its simplicity, high speed, and ease of sample preparation make it also suitable for enzymatic and clinical studies.
The technique of high pressure liquid chromatography (LC) offers many advantages in the assay of certain classes of compounds: speed, sensitivity, selectivity, and ease of sample preparation ( I ). Excellent instruments are available commercially which can deliver a single solvent to a chromatographic column a t pressures up to 3-6000 psi and monitor the effluent a t a single wavelength in a low dead volume cell. For many purposes, operation a t slightly lower pressures (up to 1500 psi) allows the use of a much less expensive pump of the pressurized tank type. If a recorder and detector are available, only columns, a flow cell, and one of these pumps would be required for a simple LC system. Other "home made" devices for LC have been described (2-8) but they are either specialized or require extensive machining. This pump is constructed of stainless
steel parts available as stock items from valve and fitting retailers, and requires less than an afternoon to assemble. The apparatus has been used in this laboratory for the trace analysis in tissues of several amino acids of neurochemical interest: 5-hydroxytryptophan, taurine, and yaminobutyric acid (GABA). These compounds illustrate the advantages of LC: they can be assayed in tissue with a 10-100 times increase in sensitivity and speed over conventional fluorimetric or absorbance methods, and the assays of these compounds in biological material require no sample preparations other than precipitation of proteins.
EXPERIMENTAL Pumping System. T h e pump in its simplest form (Figure 1) consists of a pressurized solvent tank with the necessary valves and fittings. A parts list is given in the appendix for this design, as well as for a more sophisticated 2-tank version (Figure 2). A regulator provides nitrogen to the top of the solvent reservoir. A 3-way valve a t the bottom either stops the flow of solvent or directs it to a drain or to a sample injection device. T h e injectors are available commercially, or can be made from a Swagelok Tee with a %-inch 0.d. chromatographic column in one branch of the Tee, and a Kel F insert in the other branch to serve as a needle guide and septum retainer (Figure 3a). A Teflon insert inside the Tee accepts the syringe needle, and restricts the dead volume to 5 p l (or less, if desired). T h e length of the needle guide determines whether delivery of the sample is off-column or directly into the packing. If glass columns are used they are connected with 32ga Teflon tubing (0.010-inch i.d., 0.027-inch 0.d.). A short piece of '/d-inch diameter Teflon rod drilled out with a No. 72 drill (0.025 inch) holds the tubing in the injector Tee. T h e tubing is stretched to reduce its diameter, cut, and then the thin portion is inserted into the rod, and pulled through until the unstretched tubing emerges. Excess tubing is cut off flush with the end of the rod. This type of friction-fit adapter (which is leak proof to 500 psi) is also used in the flow cell
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
375
e3 A
Figure 3. ( A ) Septum injector. (B)Fluorescence flow cell, cell holder, and tubing fittings
Figure 1. Pneumatic pump for high pressure liquid chromatography
described below and to adapt 32ga tubing to the Altex or LDC connectors or Tees used post-column. Molded adapters are available from Durrum Instrument Co., but sturdier machined fittings can be made if a lathe is available. Construction details for the injector, adapters and flow cell are available on request. T o fill the tank in Figure 1, the nut on the tubing connector a t the top of the tank is loosened, When the tank is depressurized, the tubing is disconnected, and a piece of Teflon or PVC tubing is connected between the tank and a vacuum source. After evacuating the tank, the tub-
*Sam 1
2
Figure 2. Two-solvent pneumatic pump
376
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
ing is pinched off, removed from the vacuum source, and immersed in the solvent so that the solvent is sucked back into the tank. T h e solvent tank is then repressurized. T h e pump in Figure 2 avoids disassembly by the use of check valves. T o fill the tanks, the top valve is turned to “vent” in order to evacuate the tank. When the middle valve is turned to the “draidfill” position, solvent is drawn through the “fill” check valve into the tank. After the middle valve is turned to the “of!”” position, the top valve is turned to the “pressurize’’ position. T o drain the tank, the pressure is reduced to 400-600 psi, and the middle valve turned slowly to “drain/fill”. The “fill” check valve closes, and the solvent is directed to the drain. Gas which dissolves in the solvent may interfere with the detector because of bubble formation. This problem can be reduced by minimizing the surface area with a floating barrier or by replacing the tank with a coil of tubing. The gas barrier consists of 1-cm squares of polyethylene (density = 0.95), cut from a wash bottle and dropped into the tank. Detection System. The detection of these amino acids requires the addition of a reagent to the chromatographic stream between
STANDARDS (25nmoll
TAURINE
w
0
-
0
-
z w
R A T PINEAL
i
v)
W
K
0
3
-
CYSTEIC
PHOSPHOETWNOL-
u
the column and the detector. In the present experiments an infusion pump (Harvard Apparatus) with a 50-ml Teflon gas-tight syringe was used (Hamilton). A more stable flow could probably be achieved with another pressurized tank (60 psi), a ball type flowmeter and a very fine metering valve. However, the infusion pump is preferable if corrosive solutions (such as concd. HC104) are to be used. If conventional spectrophotometers and spectrofluorometers are to be used as LC detectors, low dead volume (10-100 p1) flow cells are required, but are not available for many instruments. For monitoring fluorescence in an Aminco-Bowman and Aminco SPF 125 fluorometer the 30-p1 cell in Figure 3b was constructed. I t consists of 2-mm i.d., 4-mm 0.d. synthetic quartz tubing (SGA Scientific) with Teflon plugs drilled (No. 72 drill) to accept 32-gauge Teflon tubing. The cell is held in the light path with a black plastic block 12.5-mm square with a hole drilled out to accept the tube, and 1-mm slits cut with a saw; 2-mm slits would probably be preferable. The seal between the Teflon plugs and the cell can be made leak-tight to 500 psi if the plugs and cell are compressed with a screw. For operation a t 25 psi or less, the plugs can be held in place with heat shrinkable PVC tubing. Teflon-quartz flow cells for spectrophotometers can be constructed with a design similar to that used by Jentoft and Gouw ( 4 ) . Taurine and GABA Assay. Amino acids can be readily detected with o-phthalaldehyde (9-12). The effluent was mixed in a Tee joint (Altex) with a solution consisting of 100 mg o-phthalaldehyde dissolved in 1 ml ethanol, 100 pl 2-mercaptoethanol and 50 ml 0.5 M sodium borate buffer p H 10.3, pumped a t 0.02 ml/min. Before reaching the fluorometer, an approximately 15-s delay was allowed for reaction by the use of a 1.5-m coil of 32-gauge Teflon tubing. T h e putative neurotransmitter taurine (2-aminoethane sulfonic acid) can be readily separated from other amino acids on a 25-cm X 2.1-mm i.d. column of Aminex A-5 (Bio-Rad) a t room temperature with a mobile phase of 0.1 M NaC104 (adjusted to pH 2.2 with HC104) pumped a t 1000 psi (0.4 ml/min). At this pH, the only amino acids which are eluted rapidly are those containing a relatively strongly acidic group, such as sulfonate, sulfinate, or phosphate. The flow cell of Figure 3b was used in an Aminco SPF-125 spectrofluorometer with excitation set a t 330 nm and emission a t 450 nm, with 4-mm slits in the monochromators. The high sensitivity of the detection system allows the measurement of taurine in a single rat pineal gland. The pineal (1.9 mg) was homogenized in 50 p1 of 0.4 N HC104 in a 1.5-ml Eppendorf plastic centrifuge tube using a pestle cast of epoxy resin in a similar tube. After centrifugation, 5 p1 of the supernatant was injected into the chromatograph. GARA can be readily separated at a single p H from interfering compounds on a cation exchange resin since its pK, (4.0) is higher than that of the a-amino acids (pK. 1.9-2.3). At p H 4.5, acidic and neutral a-amino acids elute near the void volume, while GABA is retained, and is completely resolved from aromatic and basic amino acids. For this separation, the solvent (0.3 M NaC104-0.1 M sodium acetate plus acetic acid to adjust the pH to 4.5) was pumped a t 1000 psi (0.4 ml/min) through the above column a t 55 OC. The effluent was mixed with the o-phthalaldehyde solution described above, pumped a t 0.1 ml/min. An Aminco Bowman spectrofluorometer was used for these assays, with excitation a t 330 nm and emission a t 450 mm.
The high sensitivity of the assay allows measurement of GABA in the small amounts of tissue available for neurochemical study at the level of discrete nuclei. Rat substantia nigra was removed as described previously (13).The tissue, 24 pg protein, was homogenized in 100 pl 0.4 N HC104. The extract ( 5 pl) was injected directly into the chromatograph. Glutamic Acid Decarboxylase Assay. The enzyme which forms GABA from glutamate is glutamic acid decarboxylase. Measurement of the enzyme by LC is a t least as sensitive as radiometric techniques but more specific since only GABA formation is determined, not glutamate breakdown. For assay, substantia nigra from a single rat was homogenized in 100 pl sodium phosphate buffer, pH 6.5; 20 g1 of the homogenate was incubated a t 37 "C with 10 pl of a solution containing 30 mM Na L glutamate, 1.5 mM pyridoxal phosphate, and 150 mM sodium phosphate buffer, p H 6.5. After 30-min incubation, the enzymatic reaction was stopped by the addition of 10 p10.4 N HC104. A blank was prepared identically except that the HC104 was added to denature the enzyme before beginning the incubation. For assay, 5-pl aliquots were chromatographed. Since GABA need not be separated from the traces of tyrosine and phenylalanine present, the p H of the eluting buffer was raised to 5 to reduce the time required for elution (2.5 rnin). 5-Hydroxytryptophan Assay. Although 5-hydroxytryptophan does not occur in measureable amounts in the brains of normal animals, it accumulates when its catabolism is blocked with a decarboxylase inhibitor. The rate of accumulation is an indirect measure of the turnover of the neurotransmitter 5-hydroxytryptamine (14). For chromatography, the same conditions were used as for the assay of GABA (55 "C, pH 5.0, 0.4 M Na+). For detection, a filter fluorometer was used (Farrand ratio type in the non-ratio mode) with a 300-nm interference filter in the primary position and as a secondary filter, a Corning type 3-69 sharp cutoff filter. For maximum sensitivity, filter instruments are preferable to grating instruments such as the Aminco Bowman, but the latter allows wavelength scans to be made when relatively large amounts of samples are injected. The column effluent was mixed with concd HC104 pumped at 0.2 ml/min in order to take advantage of the acid-induced shift in emission of 5-HTP (from 360 nm at pH 5 to 550 nm in 3 M acid). This technique has been used for the measurement of tryptophan hydroxylase in vitro ( I s ) ,where 10 pmol of 5-hydroxytryptophan must be measured in the presence of a thousand-fold excess of tryptophan.
RESULTS AND DISCUSSION The pressurized tank type pump has proved convenient and reliable in 6-mo daily use. No interference from dissolved gas has occurred with the aqueous solutions used while operating for up to 8 hr a t 1500 psi. Taurine was rapidly separated from other strongly acidic amino acids ( t = 2.6 min) (Figure 4). The first dicarboxylic amino acid to be eluted (aspartate) came off a t 55 min as a very broad disturbance of the base line. Taurine was readily detected in the pineal, much lower amounts of phosphoethanolamine and hypotaurine were seen. Based on peak height, 2.0 nmol taurine was present, corresponding to 10 pmol/g wet wt. Other amino acids were essentially never eluted. In practice, tissues samples can be repeatedly injected until the base line becomes distorted, without regenerating the column after each sample. Then. the p H 2.2 solution can be allowed to run until the base line flattens (about an hour) and then more samples injected. Alternatively, the column can be regenerated after each group of samples by washing with 0.1 M NaOH for 5 min, and then equilibrating the column with pH 2 . 2 solution of sodium formate and formic acid. T h e elution of GABA t o o k 5 min a t pH 4.5; tryptophan and the basic amino acids were more strongly retained (Figure 5 ) . Fifty pmol gave a signalhoke ratio of 10.Peak height was proportioned to sample size to the largest amount tested (5 nmol). The GABA peak was readily detectable in substantia nigra corresponding t o 0.11 wmol/mg protein. For glutamic acid decarboxylase assay (Figure 6), the ANALYTICAL CHEMISTRY, VOL. 48,
NO. 2,
FEBRUARY 1976
377
1
GLY SER ALA THR V A L GLUNH?
STANDARDS
LEU ASP"?
0 25
ILEU MET CY s
ERAINSTEM icontroll
nmol
m
o
R A T SUBSTANTIA N I G R A
'
1
0
" " 1
1
5
IO
f i '
'
1
'
1 "
'
I5
1
1
1
20
1
25
MINUTES
STANDARDS
Separation of GABA from other amino acids, pH 4.5, 55 detection with c-phthalaldehyde
Figure 5. OC,
IlL! HOMCGENATE
AC D i F l E D D HOMOGENATE
'
- u C
'
i
3
0
1
2
3
IRAINSTEY
(Con t r o l l
BRAINSTEY
(Ro 446021
I r r c l GABA
-
0
1
2
- - -
3
0 2 4 6 810
MINUTES
Figure
(MINI
Figure 7. Separation of 5-hydroxytryptophan from tissue, pH 5.0, 55 OC. Detection by native fluorescence in strong acid at >520 nm. Excitation. 300 nm
'
'
0-0
-0
T I M E
I "
ERAINSTEM ( R o 44602)
6. Glutamate decarboxylase in rat substantia nigra, pH 5.0,
55 OC, detection with c-phthalaldehyde
0246810 T I M E (MINI
0246810
Separation of tryptophan and 5-hydroxytryptophan, pH 5.0, 55 O C , detection by native fluorescence at pH 5.0 at 360 nm. ExcitaFigure 8.
tion, 280 nm amount of endogenous GABA in the tissue extract is substracted from the amount formed during the incubation. The activity of the enzyme sample (0.5 pmol/mg prot/l5 min) was sufficient to increase the GABA content of the extract about 6-fold. Figure 7 shows the separation of 50 pmol5-hydroxytryptophan from 500 pmol tryptophan, and the chromatograms of extracts of a brainstem from an untreated rat, and of a rat treated with the decarboxylase inhibitor Ro 4/4602 (800 mg/kg, i.p., 45 min before decapitation). The brainstems were homogenized in 4 vol0.4 N HC104 and centrifuged; 50 gl were injected for assay by the stop-flow technique. The sample from the treated animal contained 17 pmol 5-hydroxytryptophan (1.6 nmol/g wet wt, assuming 60% water content of the tissue) while it was undetectable in brain378
stem of the untreated animal. The sensitivity (signal/noise = 10 for 10 pmol) of this technique is great enough to allow determination of 5-hydroxytryptophan accumulation in small nuclei (such as the dorsal raphe) which have high tryptophan hydroxylase activity, and in large areas such as the hippocampus or corpus striatum where large amounts of tissue are available. Peak height was proportional to sample size up to a t least 5 nmol. Confirmation of the identity of the apparent 5-hydroxytryptophan peak is possible by leaving the pH of the stream in the detector the same as the mobile phase (pH 5.0) and changing the filters: primary, 280 nm, interference; and secondary, a Corning 7-60 bandpass (360 nm). Aliquots of the same tissue samples were injected (Figure 8). In the brainstem of the treated
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
rat, a peak corresponding t o 17 pmol5-hydroxytryptophan was seen, along with a large tryptophan peak. With detection at 520 nm, a peak is seen a t 4 min which was obscured when the detection was a t 360 nm. This peak has the same retention time as 5-hydroxyindoleacetic acid, and its retention time, like that of 5-hydroxyindoleacetic acid is shifted t o 6 min by operation at p H 4.5. Although not a cation, this compound is retained by the column because of hydrophobic binding. Although other operating conditions would probably be preferable, this technique might prove useful in the measurement of 5-hydroxyindoleacetic acid in tissue. While the applications described here are primarily of neurochemical interest, the widespread applicability of the apparatus should be apparent. T h e availability of a simple reliable source of pulseless pressure allows rapid separation of many compounds, while the possibility of tailoring t h e detector to the characteristics of the compound of interest simplifies the requirements of chromatography by eliminating the detection of otherwise interfering compounds.
5 SS-42 X-F2 6 SS-401-A-2
7 SS-400-34TTF 8 ss-4CP2-1 9 1 0 SS-2HN 11 SS-2-T 1 2 SS-4-HRN-2 13 SS-200-2-2
1 4 SS-200-1-4 1 5 304-HDF4500 16 SS-4RB2
APPENDIX T h e pump in Figure 1 can be constructed for less than $200 with the following parts. All the steel fittings are available from suppliers of Swagelok fittings. Similar (and in some cases less expensive) valves and fittings are available from Hoke distributors. Plastic parts were purchased from Read Plastics. Rockville. Md. Part
No.
Stock No.
1 Matheson
3-580 2 SS-400-7-4 3 4 SS-400-1-4 5
304-HDF4-
Description
Required
High pressure regulator, 200-1500 psi Female connector, '/4-in. NPT-'/a-in. tube Tubing, stainless steel, '/?-in. 0.d. Male connector '/4-in. NPT-'/a-in. tube Tank. 500 ml
1 1
5 ft 1
SS-4-SE SS-4-HRN-2 SS-42X-F2 ss-100-1-2
Street elbow 'i4-h. NPT Reducing nipple '/4-'/s-in. NPT 3-way valve Male connector '/s-in. NPT-'/I 6-in. tube 10 Tubing, stainless steel, '/16-in.0.d. 11 SS-400-6-1 Reducing union '/4-'/16-in, tube 12 SS-400-3TTM Septum injector Male branch tee 'is-in. NPT-'/?-in. tube Teflon Rod 3/16-in.dia. Kel F Rod '/4-in. dia.
No. Stock No. 1 Matheson
3-580 2 Swagelok SS-200-7-4 3 4 SS-200-1-2
Description
High pressure regulator, 200-1500 psi Female connector 'is-in. t ube-'/n -in. NPT Tubing, stainless 'is-in. 0.d. Male connector '/s-in. tube-'/a-in. NPT
1 1 2
2 1 1
1 1
2
Reducing bushing '/4-in.-'/s-in. 4 NPT 17 SS-2ME Male elbow, '/a-in NPT 5 18 SS-2HCG Coupling, ' / a h . NPT 2 Tee, '/4-in. NPT 19 SS-4T 1 Female connector '/4-in. 20 SS-400-7-4 1 tube-'/a-in. NPT 1 Check valve, 25 psi 21 ss-4CP5-25 1 22 ss-100-1-2 Male connector '/B-in. 1 NPT-'/I 6-in. tube 1 Tubing, stainless steel '116-in. o.d., 1 f t 23 0.030-in. i.d. Reducing union '/4-in.24 SS-400-6-1 1 '/16-in.tube Filter 25 SS-2F4-7 1 26 SS-400-3TTM Septum injector: male branch tee ' / 8 -in. NPT- '14 -in. tube Teflon rod, 3/~6-in. 0.d. Kel F Rod '/A-in. 0.d. Teflon tape (Swagelok MS-STR-4) is used in all pipe connections. The steel fittings are of type 316 stainless steel, but the tank is of type 304 stainless, which is less resistant t o corrosion, and will not tolerate citrate buffers. Tanks of type 316 stainless are available from Hoke and Matheson.
ACKNOWLEDGMENT
1 1 1 1
I am grateful t o P. E. Hare for many valuable suggestions about the design of this apparatus and t o P. E. Byrne who helped with its construction.
1 ft 1 1 1 1
For the apparatus in Figure 2 , the following parts are required costing less than $400 Part
3 1
1
500
6 7 8 9
3-way ball valve Male adapter '/4-in. tube-'/s-in. NPT Female branch tee '/4-in. tube;'/4-in. NPT Check valve, 1-psi cracking pressure Polyethylene tubing '/4-in. 0.d. Nipple, 'is-in. NPT Tee, '/s-in. NPT Reducing nipple, '/4-in.-'/a-in. NPT Male elbow '/s-in. NPT-'/s -in. tube Male connector '/a-in. tube-'/4. in. NPT 500-ml tank
Required
1 1
5 ft 3
LITERATURE CITED (1) L. R . Snyder and J. J. Kirkland, "Introduction to Modern Liquid Chromatography," Wiley, New York, 1974. (2) L. R. Snyder, J. Chromatogr. Sci, 7 , 595 (1969). (3) P. E. Hare, Carnegie lnst. Yearbook, 72, 701 (1973). (4) R . E. Jentoft and T. H. Gouw, Anal. Chem., 40, 923 (1968). (5) T. E. Young and R . J. Maggs, Anal. Chim. Acta, 38, 105 (1967). (6) B. L. Karger and L. V. Berry, Anal. Chem., 44, 93 (1972). (7) A. F. Machin, C. R. Morris, and M. P. Quick, J. Chrornatogr., 72, 388 (1972). (8) B. E. Bonnelycke, J. Chromatogr., 45, 135 (1969). (9) M. Roth, Anal. Chem., 43, 880(1971). (10) M. Roth and A . Hampai, J. Chromatogr., 83, 353 (1973). (1 1) P. E. Hare, Space Life Sci., 3, 354 (1972). (12) J. R. Benson and P. E. Hare, Proc. Natl. Acad. Sci. USA, 72, 619 (1975). (13) M. Palkovits, Brain Res., 59, 449 (1973). (14) A . Carlsson, W. Kehr, M. Lindquist, T. Magnusson, and C. V. Atack, Pharm. Rev., 24, 371 (1972). (15) J. L. Meek and L. M. Neckers, Brain Res., 91, 336 (1975).
RECEIVEDfor review September 16, 1975. Accepted November 12.1975.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
379
