Oasis HLB® solid phase extraction

Oasis HLB® solid phase extraction (SPE) cartridges had already been investigated for use as sample purification step to remove hydrophobic interferences using ^H-PROG and ^^C-CHOL as tracers (see 4.2.1.2). The elution of those compounds with 80% and 100% methanol has already been shown in Figure 4-3. In order to evaluate the use of HLB cartridges in the new procedure based on ethanolic extracts, elution of ^H-PROG was examined with ethanol in potassium phosphate buffer (5 mM, pH 7.4). Rat brain extracts (5 volumes + 5 volumes from re-extraction of pellet after centrifugation, w/v) were dried and redissolved in 60 or 80% ethanol in potassium phosphate buffer (5 mM, pH 7.4, v/v), loaded onto cartridges (corresponding to 7 mg tissue/mg stationary phase) and eluted further with the same solvents. Cumulative recovery o f the radioactivity is shown in Figure 4-8.

With 80% ethanol, ^H-PROG was completely eluted within 15 volumes, with 60% within 35 volumes, thus giving an indication what volumes to apply in a purification protocol. It

can further be seen that ^H-PROG is interacting with the Oasis stationary phase in 60% ethanol, consistent with the chromatographic process. This is in contrast to the situation with 80% ethanol, where virtually no retention is observed.

Purification of the brain extracts by 60 and 80% ethanol elution through Oasis HLB® from hydrophobic interferences was evaluated by GC-MS. A total o f 20 ml 80% ethanol rat brain extracts (5 volumes + 5 volumes from re-extraction o f pellet after centrifugation) was passed through 3 cartridges (corresponding to 35 mg tissue/mg stationary phase) successively and further eluted with two volumes 80% ethanol in potassium phosphate buffer each time. Four volumes were taken off after each elution, dried down and analysed by GC-MS after derivatisation with MO and TMSI. ^H-PROG spiked samples were processed in parallel to monitor recovery. Recoveries were 100% after each step o f this procedure. High amounts of CHOL and its esters were observed after each elution, however, with no significant decrease after subsequent SPE steps (data not shown). Thus there seems to be insufficient retention o f hydrophobic substances on the Oasis HLB® cartridges due to preferential partitioning into 80% ethanol and /or the cartridges are overloaded with the contents of these brain extracts so that no interaction with the stationary phase takes place.

Next, ethanol extracts (5 volumes + 5 volumes from re-extraction of pellet after centrifugation) corresponding to 7, 14, 21 and 35 mg tissue/mg stationary phase were dried down, redissolved in 133 pl/mg stationary phase 60% ethanol in potassium phosphate buffer and passed through cartridges, followed by further elution with 200 p.l/mg stationary phase of the same solvent. Portions corresponding to 1% of the original sample were analysed by GC-MS after derivatisation. The samples containing 21 and 35 mg tissue/mg stationary phase overloaded the GC-column with the derivative of CHOL. However, CHOL was increasingly retained with higher ratios of solid phase adsorbent to brain tissue.

It was estimated in order to detect steroids of interest > 1 g tissue is needed as starting material. When extracts corresponding to 20, 12, and 8 mg tissue were injected, the corresponding signal of interfering CHOL from 1 g tissue extract in each case would obscure the chromatogram and damage the ionisation system. At 7 mg tissue/mg stationary phase CHOL was retained at an acceptable level.

4.2.2.3 Solvent partitioning

Excess lipidic material could also be removed from ethanol/acetic acid (3 % v/v) brain extracts by solvent partitioning prior to Oasis HLB© SPE. It was shown above (4.2.1.1) that isooctane partitioning 1 ; 1 (v/v) did not remove excessive amounts of polar or non­ polar free steroids from 80% or 90% methanol solutions. This was taken as basis to use the technique for extracts in 80% ethanol.

Purity of rat brain extracts after isooctane partitioning, redissolving in 60% ethanol in potassium phosphate buffer and passing through Oasis HLB® cartridges (samples corresponding to 35 mg tissue/mg stationary phase) was judged by GC-MS after separation of free steroid and steroid sulphate fractions by Oasis MAX® and is described in Section 4.2.2.4.

4.2.2.4 Separation of free and sulphate conjugated steroids by

hydrophilic/hydrophobic balance and/or ion exchange chromatography

Separation of free steroids and their sulphate esters was previously described in using SePak C l8® cartridges and differential elution [202]. For separation o f free from sulphate conjugated steroids after brain extraction with ethanol or ethanol/acetic acid, Oasis HLB® or Oasis MAX® anion exchange solid phase extraction cartridges were investigated here (after prior purification of extracts with isooctane partitioning and a solid phase extraction step with Oasis HLB®).

Oasis HLB® solid phase extraction

The separation of steroid sulphates from free steroids by differential elution from Oasis HLB® cartridges was attempted. Steroids were eluted from the cartridges with solvents at alkaline pH to keep steroid sulphates in the ionised form, ensuring maximal polarity differences. First, elution of ^H-PROG from the cartridges was further examined. ^H- PROG (1.1x10^ dpm) were added to 30 ml 40% ethanol in potassium phosphate buffer (5 mM, pH 7.4, v/v) containing 2% (v/v) ammonia. This solution was passed through an Oasis HLB® cartridge (60 mg) and after washing with 3 ml H2O, elution followed with

9 ml 100% ethanol. In the 40% ethanol loading fraction, 6.8 ± 0.5% of the radioactivity were recovered, 0.1 ± 0.02 % in the water wash and 94.6 ± 0.5% in the 100% ethanol

elution fraction (all values mean ± S.E.M., n=4). This indicated that free steroids and steroid sulphates could be separated by differential elution from Oasis HLB® cartridges. Separation was tested by spiking ethanolic rat brain extracts with PREG, DHEA and their sulphate esters. After purification by isooctane partitioning andan Oasis HLB® solid phase extraction step, eluates from the first Oasis HLB® (10 volumes, corresponding to 35 mg tissue/mg stationary phase) in 60% ethanol in potassium phosphate buffer (5 mM, pH 7.4, v/v) were diluted to 30% and loaded onto a second HLB cartridge. After a wash with 2.5 volumes of 30% ethanol, steroid sulphates were eluted with 4 volumes 40% ethanol in ammonium acetate buffer (10 mM, pH 9, v/v), free steroids with 5 volumes 60% ethanol in ammonium acetate buffer (10 mM, pH 9, v/v). The recovery of reference steroids that had been added to brain homogenates in the sulphate and free steroid fractions as monitored by GC-MS after derivatisation of both sulphates and free steroids to the MO-TMS-derivatives was as follows: DHEA 77.3%, PREG 79.1%, DHEAS 38.7%, PREGS 38.0%. There were no major interferences of hydrophobic or hydrophilic compounds in samples processed through this procedure without prior addition of reference compounds.

Separation of free steroids and steroid sulphates on Oasis HLB® was finally checked using different compounds in the free steroid and sulphate fraction. The reference steroids PREGS, DHEAS, EpiA, TESTO, 3a,5a-THPROG, PROG, 3a,5a-THDOC and CORT were fractionated in a similar procedure to above. The resulting elution profiles are shown in Figure 4-9. This experiment showed an inability o f this elution system to separate free steroids and steroid sulphate esters. Of the sulphated steroids, PREGS, which is more lipophilic than DHEAS, was incompletely eluted with 40% ethanol and carried through into the 60% ethanol fraction. The most polar of the free steroids investigated, CORT and TESTO are partially carrying over into the 40% ethanol fraction. It is doubtful that a group separation o f free and sulphate conjugated steroids on the Oasis HLB® cartridges is possible with another solvent system. In order to prevent elution o f CORT and other polar free steroids, the polarity o f the first elution solvent needs to be increased. As PREGS did not elute completely with the applied volume of 40% ethanol, it is unlikely that it and other apolar steroid sulphates could be completely eluted with a more polar solvent. Even if apolar steroid sulphates could be eluted with a large volume of a more polar solvent, this would very likely start to elute

The lack of separation of PREG and PREGS is contrary to the finding of the previous experiment. It has to be concluded that the carryover was not noticed due to the fact that free and sulphate conjugated steroids are analysed in the same way by the employed GC- MS method.

Oasis M ixed Mode Anion Exchange ®

In view o f the problem described above in obtaining a complete separation of free steroids and steroid sulphates, investigations were carried out on the Oasis Mixed Mode Anion Exchange (MAX®) polymer. This is the HLB® polymer modified to incorporate an anion exchange function (see Figure 4-1).

Oasis M AX® loading conditions

Free steroid and steroid sulphate elution were initially examined using the steroids PREG and ^H-DHEAS (diluted to a specific activity of 1.1x10* dpm/nmol with unlabelled compound). They were loaded onto the MAX columns in 30% ethanol in potassium phosphate buffer (5 mM, pH 7.4, v/v, 660 pl/mg stationary phase) and washed with the same solvent (83 pl/mg stationary phase). Recovery of ^H-PREG was 1.3% in the combined load and wash fractions, whilst recovery of ^H-DHEAS was 0.4% (means, n=2). Thus, free steroids and sulphate esters are retained on the MAX cartridges after loading and washing in pure solution of 30% ethanol in potassium phosphate buffer. An experiment was performed to see whether steroids are retained on the MAX stationary phase under these or other conditions in the presence of brain extract as matrix. The brain extracts (5 volumes in ethanol) were partitioned against isooctane and loaded onto and eluted from Oasis HLB in 60% ethanol before addition of ^H-DHEA. The HLB eluates were diluted to different concentrations o f ethanol and loaded onto MAX cartridges. Figure 4-10 shows elution o f ^H-DHEA from the cartridges after loading in these solvents.

As can be seen from Figure 4-10, considerable amounts of the polar ^H-DHEA were lost into the load and wash fractions upon loading in 25% and 30% ethanol. The lowest percentage of ethanol (2 0%) can be used as loading solvent mixture without significant

losses of free steroid. Having determined the most suitable solvent mixture to load brain extracts onto MAX cartridges, elution of free steroids was further examined.

Oasis M AX® free steroid elution

^H-PREG and ^H-DHEAS were loaded onto the MAX columns and after a wash, elution followed with 60% ethanol in ammonium acetate buffer (20 mM, pH 9, v/v, 0.33 ml/mg stationary phase). Recoveries of ^H-PREG and ^H-DHEAS were 92.2% and 0.9%, respectively. Other solvents for free steroid elution were investigated. ^H-PROG (diluted to a specific activity of 4.4x10^ dpm/nmol with unlabelled compound) was loaded onto an Oasis MAX cartridge, and after wash elution followed with either 40% ethanol in ammonium acetate buffer (20 mM, pH 7), 100% ethanol or ethyl acetate. Within 0.166 ml/mg stationary phase of 40% ethanol, 84.5% of the radioactivity were recovered, within 0.33 ml/mg stationary phase 92.4% and within 0.5 ml/mg stationary phase 94.9%. The recoveries in 0.083 ml/mg stationary phase and 0.166 ml/mg stationary phase 100% ethanol were 96.1 and 99.8%, respectively and in 0.066 ml/mg stationary phase ethyl acetate, 110.9 % (all values mean, n=2). Representative free steroids (^H-PREG and ^H- PROG) could be eluted with high recovery in one or several solvents. Elution of the steroid sulphate ^H-DHEAS is negligible in a low ionic strength solvent of medium polarity.

Steroid conjugate elution from Oasis MAX® and solvolysis o f steroid sulphate esters

Steroid sulphate elution from the MAX stationary phase was examined next after loading ^H-PREGS or ^H-DHEAS. The binding of the anions can be displaced by either high salt concentration or low pH (> 2 below pK of analyte) eluents. ^H-DHEAS elution with 60% ethanol in ammonium acetate buffer (0.25 M, pH 7, v/v) gave 52.2% recovery (mean, n=2), elution of ^H-PREGS with 60% ethanol in low pH buffers (pH 2, 3, 4 or 5, 20 mM, v/v) recoveries < 1% with 0.5ml/mg stationary phase. Elution with 60% ethanol in pyridine/acetate buffer (1 M, pH 5, v/v) or 60% ethanol in ammonium carbonate (0.25 M, v/v, 0.5ml/mg stationary phase) gave 31.8% and 71.9% recovery (mean, n=2), respectively.

In the above solvents, the steroid sulphates should be recovered intact after elution from Oasis MAX® cartridges. Determination of steroid sulphates by GC-EIMS was previously examined (see 3.2.4) and it was found that these conjugates are best analysed as derivatives of their free equivalents after cleavage of the sulphate group. Derivatisation efficiency was analysed after elution from Oasis MAX® in 60% ethanol in

ammonium carbonate and microsolvolysis. PREGS and DHEAS (1.25|Lig) in 30 ml 60% ethanol in ammonium carbonate were dried down after addition of internal standards, transferred to derivatisation tubes by 3x1 ml ethanol containing 0.5% NH3 (v/v) and

dried down again. They were then microsolvolysed and derivatised by MO-TMSI as described in Chapter 2. After analysis by GC-MS, peak area ratios to internal standards

were calculated and compared to those of equimolar amounts of corresponding free steroids to give derivatisation efficiency. The efficiency found was low compared to microsolvolysis and derivatisation of steroids dried from stock solution ( 1 0 0 pg/ml

ethanol/NHs 0.5%) and even direct derivatisation o f the steroid sulphates with MO and TMSI (see Table 4-7).

A preliminary investigation into solvent systems suitable for solvolysis was then carried out. Subsequently experiments were performed to check whether these solvents can elute steroid sulphates from MAX® cartridges and results are summarised in Table 4-7. It is established that solvolysis can occur in ethyl acetate acidified with sulphuric acid [28]. However, elution experiments with this solvent mixture of ^H-DHEAS from MAX (0.33 ml/mg stationary phase) gave low recovery (6.8%, mean, n=2).

Solvolysis in ethyl acetate can also be done after extraction of steroid sulphates from an aqueous medium. PREGS and DHEAS (1.25 pg each) were added to 8 ml 60%

ethanol/0.5 M NaCl/0 . 1 M H2SO4, a possible eluent for steroid sulphates from Oasis

MAX®. The solution was extracted into ethyl acetate (three times with 0.6 volumes) after dilution to 30% ethanol/ 0.1 M H2SO4 and addition of NaCl to 20% w/v. The

extract was dried over Na2S0 4, acidified ethyl acetate ( 2 ml) was added and incubation

allowed to proceed at 40®C for 16 h. The incubation mix was neutralised with pyridine, dried down and transferred to another tube with ethyl acetate. This solution was dried down either directly after addition of internal standards or after a water wash (0.5 volumes) before derivatisation with MO and TMSI. The reaction yielded virtually no derivatives (see Table 4-7, solvolysis with or without wash).

Solvolysis in acidified ethyl acetate containing benzene sulphonic acid (BSA) (50 mM), which was predicted to be a high affinity ligand of the MAX stationary phase cation was then investigated. PREGS and DHEAS (1.25 pg each) were incubated in 15 ml of this solution at 40“C for 16 h. After neutralisation with pyridine, the solution was taken to near dryness and the residue extracted three times with ether. After drying down and

addition of internal standards, the mixture was derivatised with 200 |il MO/pyridine for 1 h at 60°C and 100 pi TMSI for 3 h at 100”C. Derivatisation efficiency for this procedure is also shown in Table 4-7 (Solvolysis in BSA/ acidified ethyl acetate). The yield from this procedure could be further increased by incubation o f the solution in BSA/acidified ethyl acetate as above but in the presence of Na2S0 4 (Table 4-7, Solvolysis in BSA/

acidified ethyl acetate/ Na2S0 4). This last method gave the highest yield found for all the

methods that were compatible with elution from Oasis MAX® and could be accepted for further investigation.

After this preliminary examination, the recovery obtained in the above (Solvolysis in BSA/ acidified ethyl acetate/ Na2S0 4) method was confirmed in several further

experiments. The recoveries found were 81.2 ± 3.0 % and 63.0 ± 4.2 (means ± S.E.M, n=4) for DHEAS and PREGS, respectively.

Finally, this method was examined for use with HFBA-derivatisation. After incubation in BSA/ acidified ethyl acetate in the presence of Na2S0 4, drying down and ether extraction

as described above, the ether phase was partitioned with ether saturated H2O three times

to remove pyridine and other salts, dried down and incubated with 30 pi benzene and 30 pi HFBA a 60°C for 30 minutes. The relative response ratios of DHEAS and PREGS after this procedure are again shown in Table 4-7 (Solvolysis in BSA/ acidified ethyl acetate/ Na2S0 4 followed by H2O wash and derivatisation by HFBA).

The suitability o f BSA in acidified ethyl acetate for steroid sulphate ester elution from MAX® cartridges and separation from free steroids was then examined. Other known steroid conjugates are fatty acid esters and glucuronic acid ethers. Steroid fatty acid esters are more hydrophobic than their free equivalents and are most likely removed by the purification procedures (isooctane partitioning. Oasis HLB® chromatography) prior to the Oasis MAX® step. However, these compounds have been shown to not interfere with the GC-MS analysis of MO-TMS- or HFB-derivatives of free and sulphate conjugated steroids employed here (see Chapter 3). Steroid glucuronides are not known to be present in mammalian nervous tissue at present. Nevertheless, an enzyme for glucuronidation of steroids is expressed in mammalian brain (see Chapter 1) and to avoid any possible contamination, their elution from Oasis MAX® cartridges was investigated here.

As no radioactively labelled steroid glucuronides were available, elution of those compounds as well as free and sulphated steroids from Oasis MAX® was monitored by analysing urine samples of five human subjects where these compounds are expected. Urine extracts were loaded in 20% ethanol and the cartridge washed with the same solvent. Free steroids were eluted with ethyl acetate, steroid glucuronides with 60% ethanol in formate/pyridine buffer, pH 3 and after a wash with ethyl acetate, steroid sulphates with benzene sulphonic acid in acidified ethyl acetate. This is described in Appendix 2, where the successful separation o f the three steroid classes is confirmed. Comparison with traditional methods of urinary steroid separation indicated high recovery in all fractions.

Finally, to check elution of interferences by the above solvents for free steroids, rat brain extracts previously isooctane partitioned and passed through Oasis HLB® (corresponding to 35 mg tissue/mg stationary phase) were loaded onto MAX® cartridges in 20% ethanol in ammonium acetate buffer (20 mM, pH 7), and eluted with either 20 volumes 40% ethanol in ammonium acetate buffer (20 mM, pH 7) or 4 volumes ethyl acetate. This was followed in either case by 20 volumes 60% ethanol in pyridine/formate buffer (20 mM, pH 3, v/v; glucuronide fraction), 2 volumes ethyl acetate (wash) and 15 volumes benzene sulphonic acid in acidified ethyl acetate (50 mM; sulphate fraction). The free steroid and sulphate eluates were dried down, derivatised and analysed by GC-MS. Similar patterns of interferences were observed in the free and sulphate steroid fractions after elution with 40% ethanol or ethyl acetate, acceptable for brain sample analysis.

Thus ethyl acetate was used for further experiments in elution of free steroids from Oasis MAX® for practical reasons (smaller solvent volumes, solvent easier to evaporate).

4.2.2.5 Purification of HFBA-derivatives of steroids extracted and fractionated