Sample-MTA 6 . 5 E~12 MOLE/UL______
22KU7'21 ( l . « 4 b j BaC17.9x) (MS)
Tlie TRIO-2 GC-MS D ata System
In s tr u m e n t : 100 2 9 8 . 4
(a)
yjs 2 7 9 . 8 2 8 3 . 1 3 0 0 . 6 m/z 288 300 320 340 360 380 4 0 0 "420 VG LAB-BASESam ple:niA 812 FMOLEAJL?
Z2KW8'22 ( 1 . 9 3 8 ) B a (4 4 .7 x ) (MS)
100^ 2 7 9 . 8
The TRIO-2 GC-MS Data System
Instrument : y .F S 2 9 8 . 9 2 9 7 . 9 - 3 0 1 . 2 2 9 5 . 1 - 3 0 2 . 9 l i |W/z 2 8 9 3 0 0 3 2 9 3 4 0 3 6 0 3 8 0
(b)
3 94. 0 400 4 2 0F i g u r e 5.22 Electrospray mass spectra o f M TA (a) 0.06 nmol and (b) 0.007 nmol. 163
C hapter VI Conclusion:
S-Adenosyl-L-methionine (SAM) is an endogenous compound that is involved in a variety of biochemical reactions and cellular functions, such as transsulfuration, polyamine biosynthesis and méthylation of lipids, proteins, RNA and DNA. SAM and its related metabolites (eg. SAH, MTA and dc-SAM) have therefore been the target for several assays in tissues and body fluids. They are relatively polar compounds and consequently have relatively short retention times in reversed phase chromatography even if very low concentrations of organic modifiers are used in the mobile phase. All of the separations were carried out on a silica-based HPLC stationary phase. The HPLC parameters of these compounds on silica is limited by poor chromatography since they are silanophilic materials, which presented some difficulty in optimizing a unique HPLC profile for SAM and its related metabolites. The retention behaviour was therefore studied systematically on both reversed-phase and cation-exchange HPLC columns by changing the mobile phase pH and ionic strength. The optimization data for five compounds , SAM, SAH, MTA,Ado and IPDU on SUPELCOSIL™ LC- ABZ column and HYPERSIL ODS column showed that:
1. A higher selectivity and therefore a better separation was observed on the LC- ABZ column between pH 4 and pH 6. This could be a consequence of
protonation of the related compounds and the functional groups on the LC-ABZ column.
2. The capacity ratio of the MTA changed when the pH was lower than 4 on only the LC-ABZ column.
3. Very short retention times were measured on reversed-phase stationary phases for SAM as it has a positive charge at any pH value. The capacity ratio of SAM did not change significantly by varying pH on both columns.
4. Separation of SAM, SAH and Ado proved to be difficult on both reversed- phase columns used.
5. The ODS column was chosen for the reversed phase separation of the investigated compounds at pH 2.6-4.
The behaviour of SAM and its related metabolites was also studied on a PARTISIL SCX ion-exchange column. In cation-exchange chromatography, SAM had a much longer retention time and higher selectivity due to its sulphonium ion (the stable
positive charge) interacting with the SCX column within the pH range 2.9 to 6.5. The retention times of SAM, MTA and SAH varied significantly with changes in pH and mobile phase salt concentrations. However this did not significantly improve the separation of SAH from adenosine during cation-exchange chromatography. In single reversed-phase HPLC or in single cation exchange chromatography of the principal metabolites of SAM, overlapping peaks were always observed, therefore a combined reversed-phase and cation-exchange HPLC assay was developed. The two dimensional HPLC assay involved a gradient reversed-phase HPLC separation followed by cation- exchange chromatography.
Two dimensional HPLC permitted accurate detection of 0.050 nmol of SAM, MTA and dc-SAM and also 0.015 nmol of SAH, adenine and adenosine and allowed quantification of samples equivalent to 0.020 g of liver tissues for SAM, SAH, adenine and adenosine, 0.250 g liver tissues for MTA and dc-SAM, 0.3 ml of rat plasma and 7x10^ of mast cells (RBL-2H3) for SAM.
The average hepatic levels of SAM, SAH, MTA, dc-SAM, adenine (ade) and adenosine (ado) by this method, (nmol g ‘ wet weight) were found to be 35.5, 10.2, 0.27, 4.0, 49.3 and 43.3 respectively, from 24 hour starved rats. These amounts were different in the kidney e.g. 30, 14.7 and 0.23 nmol g * for SAM, SAH and MTA respectively. The accuracy and the bio-relevance of the measured endogenous concentrations of the investigated compounds (in 24 hour starved rats) were compared with the data obtained by others. The absolute values for SAM in rat livers are in good agreement with data previously published from 24 hour starved rats but the hepatic concentrations of SAH and MTA were found to be lower than previously reported which was probably attributable to the better selectivity of the two dimensional chromatographic method. The levels of SAM and its intracellular metabolites (ng/ million cells) in mast cells (RBL-2H3) were also investigated and found to be 36.9, 13.4, 17.8 and 16.2 for SAM, SAH, adenosine and adenine respectively. The recovery of SAM, SAH and MTA from sample preparation and Dowex column chromatography were 71.3%, 72% and 91.6% respectively. The recovery after the two HPLC methods was 68±10.5% for SAM, 92±9% for SAH and 83±7.8% for MTA. The average recovery for the entire procedure including the sample preparation was determined by the method of standard additions and two
dimensional HPLC was found to be 48.5% for SAM, 66.1% for SAH, 78% for MTA, 99.2% for dc-SAM and 103% for adenine and adenosine respectively. In comparison with the other published data based on preliminary clean up of the sample by Dowex column chromatography and their analysis using single HPLC in which the recovery factors of SAM, SAH and MTA were reported 98%, 40% and 57.6% respectively, the recoveries of SAM, SAH and MTA from Dowex column chromatography and the double HPLC method was satisfactory. Nevertheless the stability of SAM in pH 5-6 buffer for 35 days was also investigated and showed that, there was no significant decrease in the amount of standard SAM stored at 0°C and -20°C, only a decrease of
1.7% at -20°C and 5% in 0°C. SAM solutions kept at room temperature were decomposed during the same period of time at an approximate rate of 2.7% per day. The precision (R.S.D%) of data obtained from average values for the concentrations (nmol g ‘) of each investigated compound in rat liver for SAM, SAH, MTA, dc-SAM, adenine and adenosine were found to be 15%, 14.8%, 35%, 6.5%, 7% and 13.8% respectively. Also the reproducibility (R.S.D.%) for average values of SAM, SAH and MTA in rat kidneys were found to be 29.7%, 15.8% and 51.9%. The reported precisions from the other published methods of the bioassay of SAM and its related metabolites based on one step HPLC (ion-paired reversed-phase chromatography) or radioenzymatic procedures, were 14% and 18%, 14% and 54%, and 29% and 75% for SAM and SAH respectively. The precision obtained for SAM and SAH from mast cells (RBL-2H3) was better than those for the tissues and were 12.8% and 6% respectively, this may be due to less contamination present in cells and the use of a simpler clean up procedure to assay.
The two dimensional HPLC method proved to be a selective and a reproducible method for determination of concentrations of SAM, SAH, MTA, Ado, Ade and dc- SAM in different tissues and cultured cells. The advantage of two dimensional HPLC over the one dimensional HPLC assay was that the enhancement of the selectivity due to the different separation criteria benefitted from both reversed-phase and strong cation exchange stationary phases in a single assay. Peaks which seemed to be a single compound in reversed-phased chromatography usually produced three or more peaks in cation-exchange HPLC, suggesting that bio-assays for SAM and its related metabolites using a single HPLC method usually gave erroneous results, by
overestimating the amounts actually present.
Finally, in order to evaluate the use of mass spectrometry as a sensitive and specific HPLC detector for these compounds a number of different LC/MS techniques were investigated. The relative merits of different techniques including, Thermospray, Flow FAB and Electrospray were investigated in relation to normal UV detection. Thermospray was disappointing in terms of its sensitivity for these compounds and when the Selected Ion Monitoring (SIM) technique was used to monitor M/Z 298 for SAM and MTA and M/Z 284 for SAH, the detection limits for these compounds injected on the column were about 0.075 nmol for SAM, 1.7 nmol for SAH and 0.17 nmol for MTA respectively. Nevertheless use of an internal standard S-adenosyl ethionine (SAE) gave good reproducibility within runs with a regression coefficient of 0.9998 being obtained, and allowed better signal/noise ratios to be achieved. With flow FAB a better sensitivity for SAM, SAH and MTA was observed with detection limits of 0.020 nmol, 0.012 nmol and 0.003 nmol respectively being obtained. Electrospray (ESP) was investigated and provided detection limits of 0.012 nmol for SAM, 0.009 nmol for SAH and 0.004 nmol for MTA respectively.
These results show that a double HPLC method can serve for routine bioassays in conventional laboratories, and further LC/ESP MS may form the basis for a very specific and sensitive assay for this important class of compounds, in blood and other body fluids, where the concentrations are less than 0.2 nmol. Since ESP is a relatively new mass spectrometery technique, its further development should lead to further improvement in detection limits. This was illustrated by such recent developments as the high flow rate capabilities of a newly designed electrospray source, which can operate at a flow rate up to 1 ml min \ allowing direct coupling of conventionally packed column HPLC, without splitting of the eluent flow.
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