chromatographic methods

6O

Chromatographic methods

Fresenius J Anal Chem (1990) 337:60 - © Springer-Verlag 1990

09

Separation of some benzodiazepine derivatives on CN nano-TLC plates

T. Cserhfiti 1 and S. Olajos 2

Central Research Institute for Chemistry, Hungarian Academy of Sciences, P.O. Box 17, H-1525 Budapest, Hungary 2 National Institute for Nervous and Mental Diseases, Laboratory of Pharmacokinetics, Budapest, Hungary

Several chromatographic methods such as adsorption thin-layer chromatography [1], high performance liquid chromatography [2], and gas liquid chromatography [3] for the separation of benzodiazepine (BDZ) derivatives have already been described. The objectives of our work were to study the retention behavior of some BDZ on CN nano-plates and to develop eluent systems for their optimum separation.

Methods

HPTLC-Fertigplatten CN F2548 for Nano-DC were used without any pretreatment. The BDZ derivatives (1 = 7-amino- nitrozepam; 2 = bromazepam; 3 = uxazepam; 4 = oxazepam; 5 = lorazepam; 6 = nitrazepam; 7 = clonazepam; 8 - chlordiazepoxide; 9 = alprazolam; 10 = desmethyldiazepam; 11 = flunitrazepam; 12 = clorazepat; 13 = diazepam; 14 = prazepam; 15 = clobazam; 16 = tofisopam) were dissolved in methanol at a concentration of 1 mg/ml; 500 nl of these solutions was spotted onto the plates. Carbontetrachloride- ethylacetate mixtures served as eluents for adsorption chromatography in the concentration range of 0 - 1 0 0 vol.% ethylacetate (8 eluent mixtures). Water-methanol mixtures were applied for the reversed-phase separation in the concentration range of 0 - 8 0 vol.% methanol (5 eluent mixtures). After development the plates were dried at room temperature and the BDZ spots were detected by a Shimadzu Dual-Wavelength TLC Scanner CS-930 at 280 nm. The RM values were calculated separately for each compound: RM = log(1/Rf --1) and linear correlation was calculated between the RM value and the concentration (C vol.%) of the more strong solvent component in the eluent: RM = a + b . C.

Table 1. Parameters of linear correlations between the RM value of benzodiazepine derivatives and the ethylacetate (C1) and methanol (C2) concentration in the eluent

C o m - p o u n d No.

RM= a + b ' C 1 RM= a + b ' C 2

a - b sb r a - b Sb r

1 142.7 1.77 0.15 0.9834 115.5 1.87 0.17 0.9917 2 53.6 2.41 0.34 0.9715 170.5 2.36 0.16 0.9953 3 110.7 1.33 0.10 0.9856 168.5 2.50 0.20 0.9936 4 51.6 2.67 0.39 0.9696 167.5 2.46 0.20 0.9932 5 56.0 2.70 0.36 0.9747 208.5 2.93 0.21 0.9947 6 36.5 2.42 0.31 0.9769 194.5 2.57 0.36 0.9809 7 39.3 2.54 0.29 0.9807 210.0 2.84 0.22 0.9940 8 72.9 0.87 0.10 0.9666 202.0 2.83 0.40 0.9808 9 177.1 1 .31 0.13 0.9771 195.5 2.65 0.20 0.9942

10 79.8 3.84 0.41 0.9776 195.5 2.74 0.21 0.9941 11 23.8 2.74 0.29 0.9785 215.0 2.84 0.28 0.9904 12 73.7 3.60 0.38 0.9783 196.0 2.76 0.22 0.9940 13 6.3 2.34 0.22 0.9829 214.5 2.89 0.22 0.9940 14 13.3 5.22 0.65 0.9848 251.0 3.30 0.52 0.9878 15 66.1 2.90 0.32 0.9765 193.5 2.60 0.21 0.9933 16 51.6 3.07 0.34 0.9770 221.5 3.25 0.35 0.9888

optimal solvent composition for the separation of any pairs of BDZ can be calculated:

R f = 1/(1 + 10 al • 1 0 b l ' C ) - - l / ( 1 -}- 10 a2 ' 10 b2 'C)

where aa, bl, a2 and b2 are the parameters of linear correlations for any BDZ pairs to be separated. C is the concentration of ethylacetate or methanol in the eluent. The optimum separation occurs when the Rf value expressed by the above equation is at maximum. The equation has a maximum when its first derivative equals zero [4].

Abbreviations: B D Z = benzodiazepine; C N = cyano; H P T L C = high-performance thin-layer chromatography; T L C = thin-layer chromatography.

Results and discussion

The spot symmetry and compactness were the same for adsorption and reversed-phase system. However, the differences in the retention characteristics of BDZ derivatives were higher in adsorptive than in reversed-phase chromatographic mode. This finding indicates that the use of CN plates is more advantageous in pure organic than in aqueous eluent mixtures. From the parameters of the linear correlations compiled in Table 1, the

References

1. Wouters I, Roets E, Hoogmartens J (1979) J Chromatogr 179:381-389

2. Mehta AC (1984) Talanta 3 1 : 1 - 8 3. Drouet-Coassolo C, Aubert CP, Coassolo P, Cano JP (1989)

J Chromatogr 487 :295- 311 4. Cserhfiti T, Somogyi A (1988) J Chromatogr 4 4 6 : 1 7 -

22

Fresenius J Anal Chem (1990) 337:6t - 6 2 - © Springer-Verlag 1990

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10

High-performance liquid chromatographic investigations on the formation of coproporphyrin isomers II and IV in human urine

K. Jacob, E. Egeler, and P. Luppa

Institut ftir Klinische Chemic (Direktor: Prof. Dr. D. Seidel), Klinikum Groghadern der Universit/it Miinchen, Marchioninistrasse 15, D-8000 M/inchen 70, Federal Republic of Germany

Porphyrin isomers of the series I and III are normally present in biological materials. Recently, we detected for the first time small amounts of the atypical coproporphyrin isomers II and IV in urine of patients with acute intermittent porphyria [2, 3] as well as in urine of healthy subjects [4]. In this study we describe an improved sample preparation technique for the simultaneous high-performance liquid chromatographic (HPLC) analysis of the coproporphyrin isomers I - IV in human urine. This method was applied to investigate the in viv0 formation of the atypical coproporphyrins II and IV under various intravesical pH values.

Materials and methods

(9

© (9

0)

_2 cD rr"

Preparation of urine samples. 50-ml samples of freshly excreted o urine (pH 5.0 to 7.6) were oxidized with 50 mg iodine for 10 min at room temperature. After addition of 50 ml 3.7 mol/1 H3PO4, o the porphyrins were adsorbed on Sep-Pak Cls cartridges (Mill±pore, Eschborn, FRG), eluted with methanol/acetone o z (1 : 1, v/v) , and concentrated under vacuum. The residue was = dissolved in aqueous sodium acetate, brought to pH 3.5 with - acetic acid, and adsorbed on 200 mg talc. After elution with 10ml methanol/H2SO4 00:1 , v/v), the eluate was diluted tenfold with water, and then adsorbed on Sep-Pak as described above. For HPLC analysis, the isolated porphyrins were dissolved in methanolic tetrabutylammonium phosphate (pH 7.2).

HPLC separation. The coproporphyrin isomers I - I V were separated by ±socratic ion-pair HPLC as described elsewhere [3]. The content of the individual coproporphyrin isomers was quantitated by comparing the peak area ratios with calibration mixtures of authentic specimens,


Considerable amounts of urinary porphyrins are excreted in reduced form as porphyrinogens [1]. In vitro isomerization of these unstable compounds outside the human body was pre- vented completely by oxidation of the freshly excreted urine with iodine, yielding the corresponding stable porphyrins.

Enrichment and purification of urinary coproporphyrins were performed by alternately adsorbing on reversed-phase (C1s) sorbents and on talc. The extracts were free of interfering compounds in the coproporphyrin fraction, when analysed by ±socratic ion-pair HPLC. The efficiency of this clean-up procedure was confirmed by comparing the results with a TLC purification method described previously [3]. Thus, the time- consuming preparation of porphyrin methyl esters combined with the TLC separation step could be replaced by selective solid-phase adsorption techniques. The reproducibility of the HPLC method was established with a synthetic mixture of

a

"C e3

09

1 ,.J,

I I I

IV

t I I 0 10 20

I 30 min.

I

IV II

i I I I 0 10 20 30 min.

Fig. 1 a, b. HPLC profiles of urinary coproporphyrin isomers from volunteers, a Acidic urine (pH 5.0) containing 3.1% isomer II and 5.0 % isomer IV of total coproporphyrins, b Neutral urine (pH 7.1) containing 0.1% isomer II and 0.3 % isomer IV. (Peaks: I - IV = coproporphyrin isomers I - IV)

coproporphyrin isomers, offering coefficients of variation between 1 and 4% (N = 6).

Urine samples of apparently healthy persons with different pH values (5.0 to 7.6) were analysed. The highest percentages of atypical coproporphyrins were observed in relatively acidic urines (pH 5.0 to 5.5), which contained 2.8 _+ 1.1% (X ± s, N = 14) of isomer II, and 5.5 _± 1.4% of isomer IV. Urines with pH values between 5.6 and 6.6 contained only 1.4 ± 0.6% (N = 14) of isomer II, and 3.0 ± 1.3% of isomer IV. In neutral or slightly basic urines (pH 7.0 to 7.6) the percentages of isomer II (0.1 ± 0.05%, N = 10) and isomer IV (0.3 ± 0.17%) were sharply reduced.

These findings were verified by forced in vivo shifting of the pH value of urine in volunteers. Decreasing the urinary pH

62

value by intake of large doses of ascorbic acid (up to 5 g per day) resulted in a significant increase of the isomers II and IV (Fig. 1 a). On the other hand, neutralising urine by ingestion of 10 g of sodium-potassium-hydrogen-citrate (Uralyt-U, Dr. Madaus GmbH & Co., K61n, FRG) per day diminished the atypical isomers to 0.5% and lower (Fig. 1 b).

Conclusions

In vivo formation of the atypical coproporphyrin isomers II and IV occurs through non-enzymatic isomerization of copropor- phyrinogens inside the human bladder, especially when the pH value in urine is low. This is in accordance with our results obtained from in vitro isomerization of coproporphyrinogen III [4].

Acknowledgements. This work was supported by the Hans- Fischer-Gesellschaft (Mfinchen) and by Dr. W. Braunbruck (Fribourg).

References

1. Abe K, Konaka R (1989) Clin Chem 35:1619-1622 2. Jacob K, Egeler E, Hennel B, Neumeier D (1988) Fresenius

Z Anal Chem 330: 3 8 6 - 387 3. Jacob K, Egeler E, Neumeier D, Knedel M (1989) J

Chromatogr 468 : 3 2 9 - 338 4. Jacob K, Egeler E, Hennel B, Luppa P (1989) J Clin Chem

Clin Biochem 27 : 6 5 9 - 661

Fresenius J Anal Chem (1990) 337:62-63 - © Springer-Verlag 1990

11

Determination of cysteine and cystine by HPLC

H. Birw6 and A. Hesse

Experimentelle Urologie, Urologische Universit/itsklinik Bonn, Sigmund-Freud-Strasse 25, D-5300 Bonn, Federal Republic of Germany

Introduction

Cystinuria is an inborn metabolic disorder resulting in excessive urinary excretion of the less soluble amino acid cystine. Excre- tion rates up to 12 mmol/d are reported while the solubility of cystine at normal urinary pH is about 1.3 mmol/1 [1, 2]. Thus, cystinuric patients suffer from recurrent urolithiasis.

Cystine and its monomer, cysteine, are in redox-equilibrium. Cysteine has a much higher solubility than cystine. Considering this, a precise analyses of both urinary components in cystinuric patients is important. This paper reports a method for determination of cystine and cysteine by means of high performance liquid chromatography and precolumn derivatisation of amino acids with orthophthaldialdehyde (OPA).


HPLC-system. Mobile phase. Phosphate buffer: 12.5 retool/1 H3PO,, adjusted to a pH of 7.2 with NaOH. Eluent A: 0.35% THF in phosphate-buffer, Eluent B: phosphate buffer: acetonitrile 50:50 (v/v). -- Stationary phase. Spherisorb (ODS-2, 3 g), 4 x 125 mm with a Spherisorb (ODS-2, 5 g), 4 x 10 mm guard column. - Gradient system. 2 pumps LKB 2150, controller LKB 2152, gradient mixer LKB 2152-400. -- Fluores- cence detection. Shimadzu RF 535, Ex 330 nm, Em 450 nm. - Automatic sampler. Gilson M231/401. Integrator. Shimadzu C-R2AX. - Flowrate. 1 ml/min. -- Gradient. 0 - 9 rain 0% B, 9 - 1 2 rain to 100% B, 1 2 - 1 6 rain 100% B, 1 6 - 1 9 rain to 0% B, 1 9 - 30 rain 0% B.

Sample collection. 24 h urines of cystinuric patients were collected into a sampling flask containing sulfosalicylic acid

(100 m130% SSA per 2.5 1 container) for decreasing the urinary pH immediatly to a level of 1 - 2 . This procedure prevents the rather fast oxidation of cysteine to cystine during storage.

Sample preparation. Because of the weak reaction of cysteine and cystine with OPA we developed a two step procedure for overcoming this problem. For the determination of total cysteine, the urines were treated with dithiothreitol (10 gl, 12.5 mmol/1) to reduce cystine quantitatively. After completion of this reaction, 10 gl iodoacetic acid (100 retool/l) for blocking the sulfhydryl group was added. Looking only for the cysteine content of the sample, the reduction step was omitted and iodoacetic acid was added directly to the urine. The reaction product of the procedure, S-carboxymethylcysteine (CMC), yields OPA-derivatives with normal fluorescence. This pretreatment was followed by derivatisation of the amino acids with OPA. The cystine content was then calculated by the difference of two chromatographic runs of one sample.


Under the conditions described the method shows linearity in the range of 0.5 pmol to 500 pmol on column. Analytical recovery was 97.6 + 2.1% (n = 20) for cysteine and 101.6 + 7.7% (n = 20) for cystine. Using the automatic sampler the within- run coefficient of variation was 3.45% for cysteine and 4.83% for cystine. Collecting the urine directly into SSA prevents the oxidation of cysteine to cystine up to 5 days.

Figure 1 shows chromatographic runs of a urine sample of a cystinuric patient. The cysteine content was 46.2 Stool/1 and the cystine content 764.9 ~tmol/1. The method described allows a rapid and sensitive determination of cysteine and cystine in urine. The possibility of differentiation between cysteine and cystine is extremely important in diagnostic and therapy control of cystinuria because only cystine is relatively insoluble at normal urinary pH but not cysteine. Considering this, the method allows a monitoring of a special therapy of cystinuria (high intake of ascorbic acid) changing the redox equilibrium

63

A

2 mm

!CHC 13

2ram

C

CMC

2 rain

Fig. I A - C Chromatogram of a standard A and an urine sample analysed for cysteine B and total cysteine (C; same urine like B, further diluted I + 7)

of the cysteine-cystine system into the direction of the more soluble cysteine.

Acknowledgements. This work was supported by a grant of the Dr.-Robert-Pfleger Stiftung.

References

1. Asper R, Schmucki O (1982) Cystinurietherapie mit Ascorbins/iure. Urol Int 37:91 - 109

2. Hesse A, Bach D (1982) Harnsteine; Pathobiochemie und klinisch-chemische Diagnostik. Thieme, Stuttgart

Fresenius J Anal Chem (1990) 337:63-64 -- © Springer-Verlag 1990

12

The metabolic pattern of S-carboxymethyl-L-cysteine. A new study with HPLC and a novel ex vivo carbon-13-NMR-method

D. Specht 1, C. O. Meese 2, D. Ratge 1, M. Eichelbaum 2, and H. Wisser ~

1 Abteilung fiir Klinische Chemie, Robert-Bosch- Krankenhaus, Auerbachstrasse 110, D-7000 Stuttgart 50, Federal Republic of Germany 2 Dr. Margarete Fischer-Bosch Institut fiir Klinische Pharmakologie, Auerbachstrasse 112, D-7000 Stuttgart 50, Federal Republic of Germany

Introduction

The human metabolism of the mucolytic agent S-carboxymethyl-L-cysteine (CMC) has been reported to proceed via S-oxidation and/or N-acetylation [1]. Besides S-methyl-L-cysteine (MC), the corresponding S-oxides and mercapturates have been identified. A genetically determined polymorphism of sulphoxidation has been claimed to be responsible for the in- terindividual differences observed in the metabolic pattern of CMC. Other investigators, however, found thiodiglycolic acid

(TDGA) to be the only CMC-metabolite detectable in urine [2]. In order to clarify this discrepancy, we developed two methods to monitor the metabolites which are formed in fact from CMC.

Methods and materials

The following reference compounds were synthesized in high yield and chemically pure form: S-carboxymethyl-L-cysteine sulfoxide (CMC-SO, both diastereomers), S-methyl-L-cysteine sullbxide (MC-SO, both diastereomers), N-acetyl-S-carboxymethyl-L-cysteine (N-Ac-CMC), N-acetyl-S-carboxymethyl-L- cysteine sulfoxide (N-Ac-CMC-SO, both diastereomers), N- acetyl-S-methyl-L-cysteine (N-Ac-MC), N-acetyl-S-methyl-L- cysteine sufoxide (N-Ac-MC-SO), thiodiglycolic acid (TDGA) and thiodiglycolic acid sulfoxide (TDGA-SO).

For the ~ 3 C-NMR study the stable isotope labelled analogue S-carboxy-[13C]methyl-L-cysteine was synthesized. The label is retained in all known metabolites of laC-CMC. These substances can thus be identified as to individual structure [3]. In this study the urinary pattern of CMC-metabolites after a single oral dose of 375 mg 13C-CMC was analyzed in 11 healthy volunteers following 24 h after dosing. Samples could be analyzed directly without sample preparation. The urine samples are monitored by ~3C-NMR spectroscopy, using different dilutions of the reference compounds for calibration.

The second method is a rapid, specific and sensitive HPLC procedure, which uses a modified o-phthaldialdehyde/2-mer-

64

captoethanol (OPA/ME) precolumn derivatization and reversed phase chromatography [4]. N-Acetylated reference compounds as well as urine samples were treated with acylase I (EC 3.5.1.14) for 72 h at 37°C which proved to be sufficient for complete de- acylation.

Results

Using the HPLC-method, a complete separation and quantifica- tion of all CMC-metabolites with primary amino groups could be obtained. In the 24h urine samples 7 - 2 5 % of the administered dose was found to be excreted as unchanged CMC, whereas about 3% of the dose was metabolized to CMC-SO. In these subjects no urinary MC or MC-SO could be detected. Samples pretreated with acylase did not reveal a different pattern.

Using the 13C_NMR_method we observed a more variegated metabolic pattern of CMC. In all urine samples T D G A and TDGA-SO could be characterized as the major metabolites of CMC. Besides small amounts of CMC-SO (< 2%), 1 3 - 3 2 % of the administered dose were found to be excreted as TDGA, 8--19% as TDGA-SO and 6 - 2 2 % as the unchanged dl-ug.

The results of the HPLC-method have been confirmed by the 13C-NMR method. With both methods the values of excreted CMC were in excellent agreement and no MC, MC-

SO or N-acetylated compounds could be detected in the urine samples.

Conclusions It may be concluded from this study, that the proposed genetically determined polymorphism of CMC-sulphoxidation [1] does not exist, because the formation of significant amounts of CMC-SO, MC-SO and sulfoxides of N-acetyl compounds play only a minor role in the metabolism of CMC. Instead, it could be demonstrated that the major metabolites of CMC are TDGA and TDGA-SO. Furthermore, we were unable to confirm a genetically determined polymorphism of sulphoxidation in respect to sulphoxidation of TDGA, because all subjects excreted TDGA-SO in a narrow range.

References

1. Waring RH (1980) Eur J Drug Metab Pharmacokin 5:49 2. Turnbull LB, Teng L, Kinzie JM, Pitts JE, Pinchbeck FM,

Bruce RB (1978) Xenobiotica 8:621 3. Mecse CO, Specht D, Hofmann U, Fischer P, Eichelbaum

M (1989) Eur J Clin Pharmacol 36:A151 4. Specht D, Ratge D, Kohse KP, Meese CO, Eichelbaum M,

Wisser H (1988) J Clin Chem Clin Biochem 26:769

Fresenius J Anal Chem (1990) 3 3 7 : 6 4 - 65 - © Springer-Verlag 1990

13

Specific detection of ketoses for the HPLC analysis of carbohydrates

Peter Englmaier

Institut fiir Pflanzenphysiologie der Universitfit, Althanstrasse 14, A-1090 Wien, Austria

As common detection techniques for HPLC cannot distinguish between aldoses and ketoses, their applicability to the analysis of saccharides containing ketoses, for example the kestose-type oligosaccharides and the fructans, is limited. The specific method for detection of ketoses described here may overcome this problem.

Principal considerations

In contact with ketoses, resorcine gives a purple colour when heated in an acidic solution. This reaction, already described by McRary and Slattery [1] was modified by changing the solvent and the quantitative composition of the reagent to increase accuracy [2]. In contrast to the commonly used ethanol, ethyl- eneglycolmonomethylether results in the complete suppression of degassing during the heating period, and this fact makes the reaction useful for on-line detection in a HPLC analysis, too. Oligo- and polysaccharides are quantitatively hydrolyzed on acidic treatment, and their response will be adequate to the molar proportion ofketoses liberated during the hydrolysis step.

If the reaction is used together with a conventional detector, for example a differential refractometer, the ketose-content of individual substances can be simultaneously measured.

An addition of 1 part of the reagent (1 g resorcine and 2.5 g thiourea in 1 1 solvent) and 3 parts aqueous HC1 (32%) to 1 part of the HPLC-eluate followed by a heating period of 100°C for 3 min yields the maximum sensitivity. The maximum absorption was found at a wavelength of 402 nm.

Apparatus

The need for concentrated hydrochloric acid involves considerable difficulties in the apparatus design. As only glass and teflon are fully resistant to the reaction solution, most of the commercially available post column reaction devices are not suitable.

For reagent addition, a HPLC pump was sufficient, while the hydrochloric acid required an all-glass metering piston pump (consisting of two Gilson 401-1 ml piston drives). The material for the reaction chamber and the heating coil was a glass tubing with 0.75 mm i. d., connected by teflon shrinking junctions. The registration device was a LKB 2158 flowcell photometer with a high-frequency excitation lamp at 405 nm.

Results

Table 1 shows accuracy and reproducibility data on keto- pentoses (D-ribulose and D-xylulose), D-fructose as the main

Table 1. Specific determination of ketoses and saccharides containing ketoses by use of their reaction with resorcine

65

Substance Solvent type

Aqueous solvent 40% Acetonitrile in H2O

n meanQ SD% n meanQ SD%

Monosaccharides D-Ribulose 10 1.232 1.7 10 1.215 1.9 D-Xylulose 10 1.195 1.6 10 1.192 2.0 D-Fructose 25 1.068 1.4 25 1.074 1.5 Oligo- and polysaccharides

Molar proportion of ketose

Sucrose 0.500 25 0.495 2.5 10 0.508 2.7 6-Kestose 0.667 10 0.682 2.4 10 0.678 2.8 Bifurcose 0.750 4 0.765 3.0 10 0.751 2.8 Melezitose 0.333 10 0.329 2.6 4 0.332 3.2 Raffinose 0.333 25 0.335 1.7 10 0.321 2.5 Stachyose 0.250 10 0.265 3.4 4 0.258 3.5 Inulin 0.944 25 0.942 2.1 - - -

ketohexose and some oligosaccharides of the kestose- and raffinose-type. On the reaction conditions given above, the tested ketoses react easily with resorcine, and all of them yield nearly the same molar response. The results obtained with oligosaccharides were in accordance with the expected values, indicating a quantitative hydrolysis during the reaction.

Table 1 shows the ratio Q of the detector signal between an individual component (8 lag for a single determination) and the same amount of D-mannoheptulose as the internal standard. The Q values are the mean of n independent determinations, SD% is the percentual standard deviation. A TSK G 2000 PW served as the analytical column with an aqueous eluent, with a Supelcosil LC NH2 column, the eluent was 40% acetonitrile in water. The pure substances came from various sources, mainly from Fluka, Switzerland. The inulin fraction had a mean DP (degree of polymerization) of 18.

A high reproducibility as shown in Table 1 could be found within the whole linear range from 3 to 100 nmol free ketose. On the analysis conditions described in Table 1, the detection limit is 2 nmol of a single ketose. As tested with D-glucose, the response with aldoses is lower than 0.1% of that with ketoses. Effects of the acetonitrile content in the eluate on accuracy and reproducibility of the detection could not be found.

Therefore the presented method is well suited for each kind of carbohydrate analysis by HPLC, if a selective detection of ketoses is desired.

References

1. McRary WL, Slattery MC (1945) J Biol Chem 157:161 2. Englmaier P (1987) Biochem Physiol Pflanzen 182:165

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14

2',3'-Nucleotides - analytes for sensitive chromatographic evaluation of octadecyl silicagels

G.-J. Krauss 1, S. Friebe 1, and H. Nitsehe 2

1 Biotechnikum, Abteilung Bioanalytik, Martin-Luther-UniversitS.t Halle-Wittenberg, Universitfitsring 5, DDR-4010 Halle/S, German Democratic Republic 2 Serva Feinbiochemica GmbH, Carl-Benz-Strasse, D-6900 Heidelberg, Federal Republic of Germany

Silica-based adsorbents are the most frequently used materials for high efficiency reversed phase HPLC. The success of separation depends on different surface properties of the silicagels caused by different manufacturing processes [4]. Therefore, much research activity has focused on the characterization of silica-based bonded phases concerning their ligand func- tionalities and free silanol groups.

In the present study we report on the use of a nucleotide mixture in order to test simply the selectivity of octadecyl silicagels.


Nucleotides were purchased from Serva Feinbiochemica, Heidelberg. A high performance liquid chromatograph (Abimed Gilson system, equipped with a variable UV detector) (254 nm) was used. HPLC was carried out at room temperature using columns (4.6 x 250 mm) containing different octadecyl = Si 100 reversed phases, 5 gm (Serva, Heidelberg). 0.02 tool/1 ammo- nium dihydrogenphosphate (pH 6.2) at a flow rate of 1.5 ml/ rain was used. Nucleotides were dissolved in the mobile phase.


Octadecyl silicas were tested to indicate their hydrophobic and hydrophilic selectivity. Reagents for the silanization of silicas can be mono-, di- and trifunctional chloro- and alkoxysilanes. However, free silanol groups remaining after the initial silylation process are responsible for interacting with analytes.

By using 2',3'- and 2',3'-cyclic nucleoside monophosphates a highly sensitive discrimination between the packings is possible. Retention times are given in Table 1. Retention times, peak areas and heights were highly reproducible. As shown in Table 1 distinct selectivity of supports can be observed especially in the peak pattern of purine nucleoside monophosphates. 3'-AMP and 2'-GMP or 2',3'-cyclic GMP are selective markers for the chemical structure of silica surface. T-AMP and 2',3'-cyclic AMP are eluted from all packings after a long time ( > 60 rain).

Table 1. Retention times [min] of nucleotides

Compound Reversed phase supports

1 2 3 4 5 6

3'(2')-CMP 3.7 4.5 3.8 3.8 3.5 3.7 2',3'-cCMP 4.3 4.7 4.3 4.2 4.0 4.0 3'-UMP 4.6 5.7 4.7 4.7 4.2 4.5 2'-UMP 5.4 6.9 5.4 5.4 4.9 5.2 2',3'-cUMP 5.3 6.2 5.4 5.8 4.9 5.4 3'-GMP 7.9 8.5 8.0 7.5 6.6 7.0 2'-GMP t9.0 21.2 a 20.0" 20.0 15.4 17.0 2',3'-cGMP 18.8 23.3" 19.5 a 19.5 15.7 17.8 T-AMP 22.7 24.9 a 22.0 a 20.6 16.7 18.5

I monofunctional; 2 monofunctional, endcapped; 3 difunctional; 4 difunctional, endcapped; 5 trifunctional; 6 trifunctional, endcapped; a no base line separation

Among the tested supports, monofunctional, not endcapped material give the best resolution (Table 1). This result was proved also successfully with octadecyl silicas manufactured by other firms. Contrary to this result, the same elution sequence of peaks is produced, when homologous benzenes [3] are separated on these different stationary phases.

During our studies for characterization of; plant ribo- nucleases we have used the monofunctional no r endcapped material, which allows the measurement of the formation of 2',Y-cyclic nucleoside monophosphates and 2',3'-nucleoside monophosphates resulting from the enzymatic hydrolysis of yeast RNA [1]. A preferential release of guanosine nucleotides during RNA hydrolysis could be detected [2].

In conclusion, several advantages of the HPLC technique reported here become obvious: (a) The nucleotide test mixture appears to be very sensitive to monitor the chromatographic behaviour of the reversed phase material. (b) The used conditions can also yield benchmark performance data to detect changes in chromatographic resolution under column operation during life-time of a column. (c) This described sensitive method can be an essential tool also for testing octadecyl silicas provided for preparative HPLC in the expanding biotechnological fields.

References

1. Abel S, Krauss G-J, Glund K (1988) J Chromatogr 446:187-189

2. Abel S, Krauss G-J, Glund K (1989) Biochim Biophys Acta 998 : 145-150

3. Serva reversed phase adsorbents, data sheet 298 (1988) 4. Unger KK, Lork KD (1988) Eur Chromatogr News 2 : 1 5 -

19

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Fresenius J Anal Chem (1990) 337:67 - © Springer-Verlag 1990

15

A method for assay of lactitol]mannitol ratio by anion exchange chromatography with pulsed amperometric detection

Markku T. Parviainen, Harri Kokko, Ilkka Mononen, and Pekka Pikkarainen

Department of Biochemistry, University of Kuopio, SF-70210 Kuopio, Finland

In the diseases of proximal small intestine like coeliac disease changes with villous atrophy occur in permeability and absorption of small water-soluble molecules. The urinary recovery of a small polar molecule such as a monosaccharide is reduced and that of an intermediate size such as a disaccharide increased after oral ingestion [1, 3, 4, 7]. The small intestine permeability can be studied by means of simultaneous oral administration of two probe molecules, a monosaccharide and a disaccharide, and estimation of the urinary recovery of each molecule [31. Normal cases and coeliac patients can be discriminated by expression of the sugar absorption result as a disaccharide to monosaccharide ratio.

Mannitol, a small polyhydric alcohol, is widely used as monosaccharide [1, 4, 6, 8], and cellobiose [1, 6, 8] or lactulose [4, 7] are used as a disaccharide. The analytical methods for these sugars in urine include quantitative paper chromatography [1, 7], spectrophotometric [1, 6], enzymatic [8] methods and gas- liquid chromatography (GLC) [4]. The probe molecules have been measured either separately [1, 6, 8] by two different methods or simultaneously by quantitative paper chromatography [7], or by GLC [4]. The main problem with the differential sugar tests remains with complexicity of separate assays. The sample preparation is usually rather tedious in GLC.

Recently pulsed amperometric detection (PAD) of native carbohydrate molecules in liquid chromatographic (LC) column effluent has proven to be a method of choice for analysis of different carbohydrates [2, 5]. We describe the analysis of two probe molecules, mannitol and lactitol, using anion-exchange LC and PAD at alkaline pH, and we have compared the LC results with those obtained by GLC.


We used a Dionex (Sunnyvale, CA, USA) Series 4000i System combined with a CarboPac TM PAl anion exchange chromatography column (250 m m x 4 ram, I.D.), a Dionex Pulsed Amperometric Detector (PAD), and a Varian Model 4270 integrator (Varian, Walnut Creek, CA, USA). Chromatography was performed at ambient temperature using a flow-rate of 0.8 ml/min (10.5 MPa) and a mobile phase of 0.1 mol/l NaOH-1 retool/1 acetic acid-water (6:5:89, v/v/v) for 7 rain followed by a step gradient to 0.1 tool/1 NaOH for 3 rain followed by a re- equilibration with the analytical eluant for 5 rain. After every 25 samples, a wash with 0.25 mot/1 NaOH for 10 rain was used to remove strongly retaining contaminants, followed by a re-

equilibration with the analytical eluant for 5 min. Peak detection was at following PAD detector settings: E1 = 0.05 V, E2 = 0.08 V, E3 = -0 .60 V, T1 = 540 ms, T2 : 120 ms, T3 = 420 ms.

A test solution (2 g mannitol, 5 g lactitol, 10 g glycerol, aqua purif, ad 100 ml, osmolality 1505 mosm/1, prepared by the Department of Pharmacy, Kuopio University Central Hospital) was given to 38 patients with and without histologically proven villous atrophy after over-night fasting. Urine was collected for 5 h without any additives, and stored frozen at - 2 0 ° C until analysed. A 2.0-ml sample of urine was passed through a conditioned Ca 8 Sep Pak (Waters, Milford, MA, USA) cartridge (conditioning by sequential addition of 5 ml of methanol and 5 ml of water). This eluant was used for the analysis after dilu- tion 1 + 9 in water. The standard solution containing 2 g/1 (10.98 mmol/1) of mannitol and 5 g/1 04.58 mmol/1) of lactitot in water was handled in the same way. A 20 ~tl-aliquot of the sample was injected onto the column through a Millipore 0.4 ~tm filter (Yonezawa, Japan). Each sample required a total run-time of 15 min.


A comparison of our LC method (x) for urinary lactitol/ mannitol ratio with GLC method (y) gave an equation of y = 1.148x-0.006; r = 0.96; n = 38. The specificity, linearity ( 0 - 30 retool/l), sensitivity (0.1 retool/l), recovery (99.3 % - 102.0 %) and precision (between-day CV 3 .5-7 .2% in the urinary lactitol/mannitol ratio range of 0.016 to 0.99; the ratio below 0.1 is considered normal) were all acceptable in analytical terms. When compared to GLC, the present method provides at least as sufficient alternate technique in terms of specificity and sensitivity. However, the main advantages of our method are speed and precision owing to minimal sample manipulation needed. The use of lactitol as a differential sugar absorption test probe is a new approach, and lactitol/mannitol probes may give a more sensitive measure of the small intestinal function when compared to standard cellobiose/mannitol probes.

References

1. Cobden I, Hamilton I, Rothwell J, Axon ATR (1985) Clin China Acta 148 : 53 - 62

2. Dionex Corporation (1989), Analysis of carbohydrates by anion exchange chromatography with pulsed amperometric detection. Technical Note No. 20

3. Editorial (1985) Lancet i: 2 5 6 - 258 4. Hamilton L, Hill A, Rose B, Bouchier IAD, Forsyth JS

(1986) J Pediatr Gastroenterol Nutr 6 :697-701 5. Johnson DC, Polta TZ (1986) Chromatogr Forum 1 : 3 7 -

44 6. Juby LD, Dixon MF, Axon ATR (1987) J Clin Pathol

40:714-718 7. Menzies IS, Laker MF, Pounder R, Bull J, Heyer S, Wheeler

PG, Creamer B (1979) Lancet ii : 1107 - 1109 8. Strobel S, Brydon WG, Ferguson A (1984) Gut 25: 1241-

1246

68

Fresenius J Anal Chem (1990) 337:68-69 - © Springer-Verlag 1990

16

Simultaneous separation of R,S-mephenytoin and their chiral metabolites in human plasma using HPLC with/I-cyclodextrin as ehiral stationary phase

S. B. RiJhler, Ch. Wolf, and R. W. Schmid

Laboratory of Psychoactive Drug Analysis Psychiatric University Hospital, Wfihringer Giirtel 1 8 - 20, A-1090 Vienna, Austria

Introduction

Most prescribed drugs today are containing one (or more) chiral centres and are only available as racemic mixtures [1]. This fact may not only result in differences in biological activity in man, but also in a different absorption, distribution or metabolism. For metabolic studies and to correlate drug levels in blood with their clinical efficiency, adequate analytical tools to separate enantiomeric compounds are needed. There has been a large impetus to develop efficient liquid chromatographic techniques for the separation of enantiomers in order to avoid the drawbacks of 'indirect' analytical methods [2]. 'Direct' HPLC methods with chiral stationary phases presently are the most used techniques for enantiomeric separations [3].

Since stereoselective metabolism of mephenytoin has only been studied using 'indirect' methods with GC [4], a relatively new approach to chiral recognition of R,S-mephenytoin and their main metabolites in plasma is used in this study involving the formation of an inclusion complex with silica-bonded fl-cyclodextrin [5]. Effects ofpH, methanol and the composition of the triethylamine/acetate buffer in the mobile phase and

0 Z <

a -

o O9 a ~

<

F--

the flowrate onto capacity factors, separation factors and the resolution are demonstrated.


HPLC equipment used consisted of a model 320 pump with a 802 C manometric module (Gilson, Villiers le Bel, France), a Gynkotec loop injector for sample introduction, a SPD-6A variable wavelength UV-detector (Shimadzu Europe, Duisburg, FRG) and a model CI-10B integrator (Milton Roy, Rievera Beach, FL, USA). Chromatography was done using a 250 x 4.6 mm column packed with fl-cyclodextrin bonded to silica

gel (Astec-Cyclobond, ICT, Frankfurt, FRG). To enhance UV- absorbance of R,S-mephenythoin [6] and their metabolites a post-column photochemical reaction unit ("Beam-Boost xM'', ICT, Frankfurt, FRG) was used. The reaction coil was a 15 m crocheted PTFE-tubing mounted around a tubular 8W low pressure mercury lamp with emission at 254 nm, the irradiation time at a flow-rate of 1 ml/min was 90 sec. Mobile phases consisted of methanol-water mixtures with appropriate concentrations of triethylamine-acetate buffer added. Methanol and water were of HPLC grade. Mephenytoin was a gift from Gerot, Vienna, Austria, nirvanol was obtained from Supelco, Bad Homburg, FRG.

Sample preparation. Plasma samples were deproteinized with acetonitrile (1:1), centrifuged at 10000 g and the supernatant was diluted with water (1 : 5) and 25 gl were injected.

Results and conclusion

fl-Cyclodextrin has been found to be a valuable chiral stationary phase to separate the enantiomers of mephenytoin and their main chiral metabolites simultaneously. The study of the in- fluence of pH, methanol and triethylamine/acetate content in the mobile phase and the flow-rat on capacity factors, separation

A B

5

3

I L ~ 10 20 min J) 10 20 rain 1 I

Fig. 1. A Plasma sample of a patient treated with racemic mephenytoin (Epilan), B Plasma spiked with a standard mixture of R(-)-mephenytoin (2), L-nirvanol (3), D-nirvanol (4), S(+)-mephenytoin (5). [Plasma impurity (1)]

factors and resolution was necessary to optimize the separation of the above mentioned enantiomers in plasma samples. As chromatographic conditions in Fig. 1 a buffer containing 10% MeOH in 1% triethylamine/acetate (pH 5) at a flow rate of 0.75 ml/min with absorbance-detection at 254 nm were chosen.

In plasma samples, besides of R- and S-mephenytoin, the main biological active metabolite L-nirvanol, the demethylated product of R(-)-mephenytoin could be found, whereas D- nirvanol could not be detected. In accordance to [7], our results also indicate that S(+)-mephenytoin undergoes preferential hydroxylation whereas R(-)-mephenytoin undergoes preferential demethylation. This metabolic pathway explains the absence of D-nirvanol when dosing patients with a racemic mixture of

69

mephenytoin. On the other hand D-nirvanol should appear in blood if patients are treated with racemic nirvanol. This may explain the described side-effects when racemic nirvanol had been formerly prescribed as anticonvulsant.

References

1. Dennis R (1986) Pharm Int. :246 2. Armstrong DW (1984) J Liquid Chromatogr :353-376 3. Mehta AC (1988) J Chromatogr 426:1 - 13 4. Kfipfer A, Bircher J (1979) J Pharm Exp Ther 209:190 5. The ASTEC Informer (1987) 6/1 6. Schmid R, Wolf Ch (1989) J Chromatogr 478:369 7. Kfipfer A (1979) Fed Sci Proc 38:742

Fresenius J Anal Chem (1990) 337 :69 -70 - © Springer-Verlag 1990

17

HPLC-determination of purine and pyrimidine compounds in biological samples: sample preparation and analysis

Bodo Sehertei and Wolfgang Eichler

Institut fiir Biochemie der Universitfit Heidelberg, Im Neuenheimer Feld 328, D-6900 Heidelberg, Federal Republic of Germany

Introduction

In the course of preliminary experiments we had gained some evidence that the observed physiological and morphological changes in the livers of arginine starved rats [6] were accom- panied by or due to changes in the hepatic purine:pyrimidine ratio. For further studies on that subject we decided to quantitate these substances by means of HPLC analysis. How- ever, the available systems [1, 3] had been developed for the determination of free bases and nucleosides but not nucleotides. Therefore we had to choose a specific sample preparation procedure in which the nucleotides were converted into nucleosides. Furthermore, we were interested in the concentrations of orotic acid in the samples because this pyrimidine has been claimed to be responsible for the mentioned alterations [2, 7], and in the concentrations of the redox coenzymes NAD(H) and NADP(H) because we had been able to prove mitochondrial damage by pyrimidines in vitro [5].

Since those substances had not been regarded by the published HPLC-separations we had to modify these methods according to our requirements. The results of these studies are given in this contribution.

Methods

The HPLC device by Merck/Hitachi [4] for low pressure ternary gradient formation, with a 655A spectrophotometer (12 gl flow cell) and a D-2000 Chromato Integrator was loaded via a 20 pl sample loop. The column was a LiChroCART 250-4 (LiChrospher 100 RP-18e, 5 gm). Each sample was run twice - once with detection at 254 nm, once with detection at 286 nm - taking account of the different absorption maxima of the bases.

Sample preparation 3 g of liver tissue were homogenized with 15 ml 500 mmol/1 tr iethanolamine. HCl-buffer, pH 8.0, containing 30 mmol/1

NH4C1, 5 mmol/1 e-ketoglutarate and 40 pg glutamic dehydro- genase [EC 1.4.1.3]. The homogenate was allowed to stand for 10 min and was then ultrasonicated in a water bath at room temperature (5 rain). Thereby, all the nicotinamide-di- nucleotides were converted into the acid stable oxidized form (NAD(P)+). In order to remove the macromolecules, 2 ml of the homogenate were mixed with 20 gl allopurinol (internal standard), 2 ml 1 mol/1 perchloric acid and 1 ml n-heptane (to dissolve the fats of the "fluffy layer") and centrifuged for 15 rain (ca. 20000 × g, 4°C).

2 ml of the aqueous phase were neutralized with I00 gl 5 tool/1 K2CO3 and centrifuged again (5 rain) after 15 rain on ice, 100 pl of the clear supernatant were mixed with 100 gl of 100 mol/l triethanolamine • HC1 buffer, pH 8.0, containing 2.5 gg alkaline phosphatase from calf intestine [EC 3.1.3.1], incubated for 2 h at 37 ° C, and frozen until injection.

Results

From the comparison of the internal standard yield and from "standard addition" assays it was calculated that less than 10% of the purine and pyrimidine compounds were lost during the sample preparation without exception for any compound. This loss was not so high as to interfere with the internal/external standard method [4] employed to calculate the original concentrations in the sample.

An unexpected finding was that the alkaline phosphatase treatment left NADP ÷ untouched in samples, whereas in identically treated standards only NAD ÷ was detectable. Prob- ably the high alternative substrate concentrations and the low NADP ÷ concentration protect this cosubstrate.

The obtained retention times and peak resolutions of one typical run are shown in Table 1. The correlation coefficients of calibration curves were > 0.99, the deviation between replicate injections of the same samples were below 5 %. The poor resolution of the pairs uric acid/uracil and NADP+/cytidine was compensated by the double wavelength method, the failure to separate guanine/hypoxanthine was without consequences because the latter base was quantitatively of minor importance in rat liver tissue. Typical values obtained by this method (for control animals [days 4 to I2]/arginine starved animals [days 4 to 12]/arginine refed - from day 8 - animals [days 9 to 15]) were: orotic acid (40/480/20 nmol/g), uridine (420/610/830 nmol/g), cytidine (38/7800/30 nmol/g), uracil (1.1/5.05/1.12 ~tmol/g), adenosine (95/40/57 nmol/g), guanosine (128/65/135 nmol/g), guanine, (4.5/1.3/3.9 gmol/g), adenine (330/120/70

70

Table 1. Gradient profile, retention times and resolution ofpurine and pyrimidine bases and nucleosides and nicotinamide coenzymes in HPLC-separation: solvent A: 40 mmol/1 K-phosphate buffer pH 5.8; solvent B: methanol; solvent C: water (the addition of the third component, water, was required to avoid the precipitation of phosphate at higher methanol concentrations). The gradient controller provided a linear gradient from each given ratio to the next one; flow rate was 1 ml/min

Substance Resolution Time [mini Gradient

(retention) (gradient) %A %B %C

- Start - 0 100 0 0 1 99 1 0

Orotic acid BB 3.13 cytosine BB 3.57 uric acid BV 4.70 uracil VB 4.96

uridine BB 7.48 guanine + hypoxanthine BB 8.58 xanthine BB 9.80

NADP + BV 10.30 cytidine VB 10.74 deoxycytidine BB 11.15 thymine BB 11.90 allopurinol BB 12.82 (i. s.) NAD + BB 13.74 adenine BB 14.31

guanosine BB 15.31 thymidine BB 17.40

adenosine BB 20.08

5 99 1 0

10 80 10 10

15 70 15 15

20 50 25 25

24 50 25 25

Deoxyguanosine and deoxyadenosine were omitted in this scheme. When present, they eluted well separated between 22 and 25 rain. Resolution: B = baseline, V = separation less than 75% of total height

nmol/g), NAD + (59/28/43 nmol/g), NADP + (1.1/0.55/1.3 gmol/g), and cytosine ( ~ 10 nmol/g). The values are means of 6 samples taken at different days of the assay period, the SEM never exceeded 20%.

Conclusions

A useful method is presented which allows the quantitation of purine and pyrimidine bases and nucleosides and of NADP + and NAD + in biological samples, especially in rat liver.

References

1. deArbeu RA, vanBaal JM (1982) J Chromatogr 229 : 67 - 75 2. Durschlag RP, Robinson JL (1980) J Nutr 110:816-821 3. Eells JT, Spector R (1983) Neurochem Res 8:1307-1320 4. Eichler W (1989) Biol Chem Hoppe-Seyler 370:1113-

1126 5. Eichler W, Schertel B (1988) Biol Chem Hoppe-Seyler

369:1287-1293 6. Schertel B, Eichler W (1987) Biol Chem Hoppe-Seyler

368 : 1129-1130 7. von Euler LH, Rubin RJ (1963) J Biol Chem 238:2464-

2469

71

Fresenius J Anal Chem (1990) 337:71 - 72 - © Springer-Verlag 1990

18

Determination of amino acids, monoacetyl-polyamines and free polyamines by HPLC-analysis employing pre-column derivatization with o-phthal-dialdehyde

Wolfgang Eichler*

Institut ftir Biochemie der Universit~it Heidelberg, Im Neuenheimer Feld 328, D-6900 Heidelberg, Federal Republic of Germany

Introduction

Polyamine requirement in cells greatly depends on the growth state. Polyamine concentrations are regulated on the level of biosynthesis via the regulative key enzyme ornithine decarboxyl- ase [EC 4.1,1.17] and on the level of excretion to the extracellular space, in mammalian tissues mainly as the monoacetyl- derivatives which may be regenerated to free polyamines or eliminated by urinary excretion [3]. Increased polyamine turn- over is one marker of malignant growth, e.g. cancer, and polyamines and their derivatives have been used successfully as diagnostic tools for therapy control in cancer patients [2, 3]. However, no easy-to-use analytical system has been available to determine these substances without experimental expense, e.g. temperature gradients or post column derivatization [2, 4]. Es- pecially the two isomeric monoacetyl sperm±dines have been difficult to resolve. An HPLC reversed phase separation method developed for the separation of the whole physiological set of amino acids employing pre-column derivatization with o- phthaldialdehyde (OPA) proved suitable also for the quantitation of the polyamine derivatives.

Methods

The HPLC-device by Merck/Hitachi and the derivatization procedure with OPA-reagent were as published previously [1]. After injection of the reaction product (20 btl) onto the LiChroCART 125-4 column (containing LiChrospher 100 RP-18e, 5 btm) a binary nonlinear gradient was used for elution (solvent A: 0.1 tool/1 Na-acetate buffer, pH 7.3, 9.5% methanol, 0.5% tetrahydrofurane; solvent B: methanol; gradient profile: t[min]/ %A: 0/95; 1/88; 4.5/93; 11/93; 11.5/88; 13/85; 13.5/81; 18/81; 19/72; 25/72; 26/70; 29,5/70; 30/70; 36/65; 38/65; 39/50; 42/40; 50/40; 51/15; 56/15; 57/0; 58/0; flow rate: 2ml/min; the gradient controller provided a linear gradient from each given point to the other).

Standards in the concentration range from 5 btmol/1 to I mmol/l were used to determine the retention times and the peak area ratio factor (F~s) with norvaline (norVal) and/or 1,4- diaminocyclohexane (DACH) as internal standards (C = concentration; PA = peak area; es = compound external standard; is = internal standard):

PAi~' C~s Fe s - - _ _

PAe~. Cis"

From the obtained calibration curves the correlation coefficients

* Presen t address . Minister±urn f/ir Umwelt, Raumordnung und Landwirtschaft Nordrhein-Westfalen, Schwannstrasse 3, D-4000 Dfisseldorf 30, Federal Republic of Germany

Table 1. Retention times, resolution, and correlation coefficients of the amine compounds: n = number of runs used for the calculations of means ± SEM; * = completely overlapping peak, determined in independent assays; Resol. = resolution; B = baseline separated; V = overlap of more than 25% of total peak height; Corr. = correlation coefficient: I: • 0.99; II: • 0.97; III: •0 .90; IV: •0 .75; n.d.: no concentration dependence determined; i. s. : internal standard

Compound Retention time n Resol. Corr. _+ SEM [mini

Cysteic acid 1.82 _+ 0.02 4 BB I Asp 2.05 ± 0.02 12 BB I Glu 3.16 ± 0.03 12 BB I CM-Cys 4.17 ± 0.04 4 BB I Asn 5.69 ± 0.08 12 BB I Ser 7.64 ± 0.11 12 BB I Glu-Ala 8.01 ± 0.10 4* (VV) n.d. GalN 8.33 ± 0.15 4 BV II GlcN 8.68 ± 0.12 4 VB II Gin 11.02 ± 0.18 12 BV I His 11.58 ± 0.18 12 VB I Canavanine 12.82 i 0.22 4 BB II Met-Sulfoxide 14.52 _+ 0.10 4* (BV) n.d. Met-2nd peak 14.83 ± 0.10 - (VB) n.d. Gly 15.24 ± 0.09 12 BB II Thr 15.94 _+ 0.08 12 BV I Met-Sulfone 16.38 ± 0.15 4* (VV) n.d. Cit 16.61 ± 0.10 12 VB I Arg 17.48 ± 0.11 12 BB I HomoArg 19.31 ± 0.12 4 BB n.d. fl-Ala 19.64 __ 0.13 12 BB I Tau 20.65 +_ 0.08 12 BB I Ala 21.19 ± 0.07 12 BB I 7-ABA 21.82 ± 0.09 4 BV II fi-ABA 22.08 _+ 0.09 4 VB II Tyr 23.63 i 0.10 12 BB I ethanolamine 25.73 ± 0.11 12 BB I ammonia 26.44 _+ 0.13 4 BV III e-ABA 27.20 ± 0.08 4 VB II NS-Ac-Spd 27.84 ± 0.10 8 BB II NKAcSpd 29.56 ± 0.11 8 BB II agmatine 31.66 ± 0.23 4 BB I histamine 34.36 ± 0 .20 2* (BV) n.d. Met 34.76 ± 0.14 12 (V)BB I Val 35.69 _+ 0.13 12 BV I N-Ac-Put 36.09 ± 0.11 4 VB I Trp 37,07 ± 0.16 12 BB II norVal 37.56 ± 0.09 12 BB i.s. Phe 40.25 ± 0.07 12 BB I N-Ac-Cad 40.97 ± 0.06 4 BB I Ile 41.84 __ 0.04 12 BB I Leu 42.49 ± 0.04 12 BB I Orn 44.82 + 0.05 12 BB I Lys 46.17 i 0.07 12 BB I Spd 48.50 +_ 0.10 12 BB III Put 52.11 ± 0.02 12 BB I Cad 52.64 i 0.02 12 BB I DACH 52.94 ± 0.02 12 BB I Spn 55.89 _+ 0.28 12 BB IV

72

were calculated by linear regression analysis. These "external" standard runs allowed to evaluate sample runs (also containing the internal standards) by the "internal/external standard" method with high accuracy (sa = compound sample):

PAsa Csa : ' Cis' Fes.

PAis

Results

Table 1 shows that a very efficient chromatography system has been established which allows the resolution of at least 42 of the 48 tested amino compounds (amino acids, amino sugars, and polyamine derivatives) within 1 h gradient run. The method allows quantitation of these substances in the range from 25 pmol to 500 pmol with less than 5% error at replicate injections of the same sample.

Conclusions

This optimized chromatography system can be a valuable tool for the HPLC analysis of physiological amine compounds. Es-

pecially the separation of the two isomeric N-acetyl-spermidines makes it useful for urinary metabolites control, e.g. in cancer therapy. The disadvantages of this method are the poor reproducibility and sensitivity in the determination of spermidine (Spd), spermine (spn), and N-acetyl-Spn. Probably, the non- derivatized secondary amine groups of those polyamines allow hydrophilic interactions with the silica backbone of the hydrophobic stationary phase causing a steady dispersion of the derivative under equilibrium conditions. This theory is also supported by the observation that all peaks of the diamines - lacking secondary amines - appear sharp. Stationary phases without hydrophilic residues or nonequilibrium conditions, e.g. [1], are required to handle well also those problematic compounds.

References

1. Eichler W (1989) Biol Chem Hoppe-Seyler 370: 1113-1126 2. Seiler N (1983) Meth Enzymol 9 4 : 2 5 - 2 9 3. Seller N, Bolkenius FN, Rennert OM (1981) Med Biol

59 : 3 3 4 - 346 4. Seller N, Kn6dgen B (1980) J Chromatogr 221:227-235

chromatographic methods

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