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An evaluation of the use of sep-pak C18 cartridges for the extraction of vitamin D3 and some of its metabolites from plasma and urine
J. smvid Biochem.Vol. 19,No. 3, pp. 1349-1354.1983
0022-4731/83 S3.00+0.00 Copyright(c;1983 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
AN EVALUATION OF THE USE OF SEP-PAKt Cl8 CARTRIDGES FOR THE EXTRACTION OF VITAMIN D, AND SOME OF ITS METABOLITES FROM PLASMA AND URINE C. J. RHODES, P. A. CLARIDGE, D. J. H. TRAFFORD and Steroid
Laboratory,
Department
of Chemical Pathology, The London Street, London El 2AD, UK
H. L. J. MAKIN*
Hospital
Medical
College,
Turner
(Received 15 November 1982) Cl8 cartridges for the extraction of vitamin D and some of its metabolites by studying the recovery of added tritiated secosteroids. The preparation of the cartridges, recoveries, extraction and elution with a number of solvents, effect of varying flow rates for application and elution, and the effect of increasing volumes of plasma and urine have been investigated. Two methods for the application of secosteroids present in plasma to Sep-Pak Cl8 cartridges have been examined, using methyl cyanide extracts removing precipitated protein by centrifugation, and using acidified methanolic plasma. Methyl cyanide extracts applied to Sep-Pak Cl8 cartridges and eluted with methanol or methyl cyanide gave the cleanest extracts suitable for direct HPLC. Acidified methanolic plasma, applied to Sep-Pak Cl8 cartridges and eluted with methanol or methyl cyanide gave extracts which could not be applied directly to an HPLC-further fractionation using Sep-Pak XL cartridges was necessary. Recoveries of added tritiated secosteroids using both methods were greater than 80% with the exception of vitamin D itself which was poorly recovered-methyl cyanide extraction giving only 30% recovery and use of acidified methanolic plasma giving 66% recovery.
Summary-The
use of Sep-Pak
from plasma and urine has been evaluated
INTRODUCTION
Although radioimmunoassays for some steroid hormones have been described which can be carried out directly on plasma [I] or urine, some criticisms have been levelled at their specificity [2]. Unlike such steroids, a prerequisite for the successful analysis of vitamin Dt and its metabolites in plasma or urine by any procedure, is, at the moment, their extraction from the aqueous medium pri2r to chromatography and/or quantitation. Quantitative extraction with organic solvents can be achieved and many assays use time consuming, multi-extraction procedures to obtain reasonable recoveries [3] using large volumes of organic solvents which have to be evaporated. A study of four different solvent extraction procedures prior to radioimmunoassay has been described [4]. This paper describes the evaluation of an alternative approach, using reverse-phase Sep-Pak Cl8 cartridges for the preliminary removal of vitamin D and some of its metabolites from plasma and urine. *To whom correspondence should be sent. TRegistered Trade Mark. fsystematic and trivial names of vitamin D and its derivatives used in this paper are: Vitamin D, (9,10-seco5,7,10(19),22-ergostatetraen-38-01): D,. Vitamin D, (9,10-seco-5,7,10(19)-cholestatrien-3~-o~): D,. 25-Hyhroxyvitamin D,: 25-OHD,. 25-Hydroxyvitamin D,: 25-OHD,. 24.25-dihvdroxvvitamin D,: 24,25(OH),D,. lcc,25-diiydroxyvitamin D;: 1,25(0H)&. The term b is used when it is not required to distinguish between Dz and D,.
Although Sep-Pak Cl8 cartridges have been used for the extraction of steroid hormone metabolites from urine [5] and plasma [6] and for the extraction [7, 81 and fractionation [7] of 25-hydroxyvitamin D from plasma prior to high-performance liquid chromatography (HPLC), no proper evaluation of their use for the extraction of vitamin D and its metabolites has previously been published. MATERIALS AND METHODS Reagents
25 Ci/mmol), [ 1,2-‘HIvitamin D, act. (SP. act.: 25_hydroxy[23,24-‘HIvitamin D, (sp. 102 Ci/mmol), 24,25-dihydroxy[23,24-3H]vitamin D, act.: 77 Ci/mmol) and Ir,25-dihydroxy(sp. [23,24-3H]vitamin D3 (sp. act.: 120 Ci/mmol) were all obtained from Amersham International PLC. (Amersham, Bucks., UK) and were found to be radiochemically pure ( > 98%) on receipt but were re-purified immediately prior to use by highperformance liquid chromatography (HPLC). When added to plasma or urine, irrespective of volume, for recovery experiments, 20,000 dpm of each secosteroid was used unless otherwise stated. Other reagents used, unless otherwise specified, were the highest grade available (usually Analytical Reagent) from BDH Chemicals Ltd (Poole, Dorset, UK) and were used as supplied without further purification. Solvents used for HPLC were obtained from Rathburn Chemicals Ltd. (Peebleshire, UK) and HPLC was
I349
carried out as described previously [8,9]. HPLC solvents were degassed before use. Sep-Pak Cl8 and SIL cartridges were purchased from Waters Associates (UK) Ltd (Northwich. Cheshire, UK). Liquid scintillation counting was carried out using an fntertechnique model SL3000 (Kontron Intertechnique Ltd. St. Albans, Herts., UK) and 5 ml of NE250 liquid scintillation counting fluid. At least 1000 counts were collected. Unquenched tritium efficiency was around SW!<,falling to around 40”,/, for the most severely quenched samples, and the background count rate was approx 30cpm. Quench correction, where necessary, was carried out using an external added standard (jH hexadecane, Amersham International Ltd).
Sep-Pak Cl8 cartridges were prepared before use by successively washing each cartridge with 10 ml of methanol and 10ml of water. Radioactive secosteroids were added to aliquots of urine or plasma which, after incubation at 37°C for 15 min and room temperature for 45 min, were extracted by shaking on a vortex mixer for 60s with an equal volume of methyl cyanide. In the case of plasma, the precipitated protein was removed by centrifugation (15OOg for 10 min), and the extract was then applied rapidly (flow rate > 24 ml/min) to a Sep-Pak cartridge. The cartridge was then with 3ml of washed methanol-water (70:30, v/v) and the vitamin D metabolites eluted by slowly (flow rate < 24 ml/min) passing through the cartridge, 4 ml of methyl cyanide. In cases where the volume of plasma or urine was greater than 5 ml, 0.5 g of ammonium sulphate per ml of original sample was added to the methyl cyanide extract before it was applied to the Sep. Pak Cl8 cartridge. The mixture was shaken and centrifuged (I 500g for 10 min). The clear upper methyl cyanide layer was removed, evaporated to dryness under a gentle stream of nitrogen at 37”C, taken up again in 10 m! of methyl cyanide-water (I : 1 v/v) and then applied to the Sep-Pak cartridge, which was washed Table I. Recoveries of added tritiated secosteroids from plasma (2 ml) and urine (2 ml), applying methyl cyanide extracts to Sep-Pak Cl8 cartridges Secosteroid D, 25OHD, 24,25(OH)zDT 1.25(OH),D,
“i, Recovery (mean f SD, n = 5) Urine Plasma 73.5 + 10.9 91.7 $- 3.2 79.9 * 5.2 88.6 * 7.1
6.9 i 84.9 + 83.9 * 82.6 k
3.3* 9.8 14.0 3.9
*Using silanised Sep-Pak cartridges, this recovery can be increased to give a quantitative recovery, after dUtiOn from the Sep-Pak cartridge, of the total amount extracted into the methyl cyanide layer (26.6 + 7.10/,). Use of silanised Sep-Pak cartridges does not affect the recoveries of other secosteroids examined. Recoveries of all secosteroids, other than D, in the methyl cyanide extract was quantitative (Plasma 102.4 k 5.3, n = 15; urine 101.6 f 9.7, n = L5). Details of extraction and elution procedures are given in the “Method” section in the text.
with methanol-water as descibed above.
and eluted w~tb methyl c’!;mi _
RESU
f.‘l’S
Preparation c!f Sep -Pa.4 curtridgw Sep-Pak Cl 8 cartridges were prepared before use as recommended by the manufacturers by passing methanol and water through the cartridge ;ts dcscribed in the Method section above. Experiments using tritiated 2S-OHD,, 24.25(OH),D,. and 1,25(OH),D, showed that the length of time between preparation and use was not crucial since similar recoveries of added tritiated secosteroids were ohtained using cartridges prepared immediately and up to 4 h before use.
Recovery studies Tritiated secosteroids were added to plasma and urine which were processed as described in the Method section above. The final extract was counted on a scintillation counter to assess recovery. HPLC was carried out on some of the extracts and in no case was the radioactivity not eluted from the HPLC with the appropriate standard secosteroid. Table I gives the results of these experiments. In an attempt to increase the recoveries of added secosteroids. SepPak C 18 cartridges were “silanised” by soaking in I ‘I(, dimethyldichlorisilane in toluene for I h. washed with methanol and finally prepared as described above. Silanisation of Sep-Pak Cl8 cartridges had no ctfect on the recovery of any of the secosteroids studied except for vitamin D,. the recovery of which, in this experiment, was increased from a mean of 6.7”,, to a mean of 3 I .7’!$
Extraction md elthrm studies Tritiated secosteroids were added to 2 ml aliyuots of plasma and extracted with a variety of solvents (methanol, ethanol, acetone, methyl cyanide). Results were similar to those reported previously [8]-methyl cyanide consistently gave the highest recovery of all the steroids examined also giving the cleanest extracts as judged by HPLC analysis. There seemed to be little difference, however, between methanol and methyl cyanide as eluting solvents: both giving clean extracts and similar recoveries. All tritiated secosteroids examined were eluted completely from the cartridge with 4mI of either methanol or methyl cyanide. In an attempt to fractionate the secosteroids, methyl cyanide extracts of plasma to which tritiated secosteroids had been added were applied to cartridges which were then etuted with methanol- water mixtures. Poor elution profiles, similar to those reported previously [7], were obtained with inadequate resolution between the secosteroids.
Effect of vwying ,fh
rutrs of’ upplicutiorz and drtfim
Considerable variation in recovery was initially experienced, so the speed of application of methyl
1351
Vitamin D, and metabolite extraction by Sep-Pak Table 2. Effect of varying flow rates of application to and elution from Sep-Pak Cl8 cartridges using the methyl cyanide extraction method Flow rate (ml/min) 144
24 19 12 2
% Recovery of added ‘H 25-OHD, Application* Elution 83.6 89.9 54.3 50.0 36.5
49.5 84.8 80.0 85.6 81.2
*Methyl cyanide extracts were applied at the different flow rates indicated (and washed and eluted at a flow rate of 7 ml/min) or applied at 30ml/min and washed and eluted at the flow rates indicated. Values recorded are the means of duplicate experiments. Details of elution and extraction are given in the “Method” section.
cyanide extracts to the Sep-Pak cartridge and the subsequent rate of elution was examined. Cartridges were attached to a peristaltic pump (AA11 AutoAnalyser), which could pump solvent through at different flow rates (2-144 ml/min). The recovery of tritiated 25-OHD, obtained when methyl cyanide extracts of plasma were applied to and eluted from the Sep-Pak cartridge at different flow rates were measured. The highest recoveries were obtained when extracts were applied rapidly (24144 ml/min) and were eluted slowly ( < 24 ml/min). Details of recoveries at different flow rates are given in Table 2. Effect of increasing volumes of plasma and urine Large volumes (> 5 ml) of plasma and urine, extracted with the appropriate volume of methyl cyanide gave rise to low recoveries of added tritiated 25-OHD, from the Sep-Pak cartridge. Evaporating the methyl cyanide extract to dryness, which is a lengthy procedure because of the large amount of water present, and subsequent application of the dried extract, re-dissolved in 10 ml methylcyanide: water (1: 1, v/v) to the cartridge did partially improve recovery but only to around 80% of that achieved with small volumes of plasma ( < 5 ml). If ammonium sulphate (0.5g per ml of sample) was added to the methyl cyanide extract before application to the Sep-Pak Cl8 cartridge, shaken and then centrifuged, two immiscible layers were obtained. The clear upper layer contained methyl cyanide which was free of water and could thus be more easily evaporated. If this dried extract was re-dissolved in 10 ml methyl cyanide-water (1: 1, v/v) and re-applied to the cartridge, recoveries were completely restored to that obtained using small volumes of plasma and urine. Table 3 shows the effect on the recovery of added ‘H 25-OHD,, of extracting increasing volumes of plasma and applying the extract to Sep-Pak cartridges before and after treatment with ammonium sulphate. Steroid hormone metabolites have been recovered from urine by passing it directly through Sep-Pak Cl8 cartridges[5] and eluting the adsorbed steroids
with methanol. Increasing volumes of urine (5-lOOml), to which ‘H 25-OHD, had been added, were passed through Sep-Pak cartridges, which were then washed with methanol-water and eluted with methyl cyanide as described in the Methods section above. As the volume of urine used increased the recovery of tritiated 25-OHD, dropped (from 91.7% using 2 ml to 14.6% using 100 ml). However, if the methyl cyanide extract of urine was treated with ammonium sulphate as described for plasma in the preceeding paragraph, added tritiated 25-OHD, could be recovered from large volumes of urine (20ml of urine applied directly gave a recovery of 34.9% but when extracted, and treated with ammonium sulphate, recovery was restored to that achieved using small volumes of urine). Examination tridges
of other procedures using Sep -Pak car -
Other procedures for the extraction of 25-OHD [7], oestrogens [lo] and steroids [6] from plasma using reverse-phase Sep-Pak cartridges have been described. These procedures do not use methyl cyanide extraction but instead directly apply to the Sep-Pak column acidified plasma [6, lo] or acidified methanolic plasma [7]. All three procedures were studied using tritiated 25-OHD, added to 5 ml of plasma. Only the method of Dabek et af[7] using acidified methanolic plasma gave reasonable recoveries, the other two procedures, using acidified plasma without methanol, gave recoveries of less than 10% of added 25-OHD,. The method of Dabek et al.[7] was therefore studied using all four tritiated secosteroids. Each secosteroid was added to 3 ml aliquots of plasma and after incubation, the plasma was treated as described by Dabek et a1.[7]. This procedure involves acidifing the plasma by addition of 4ml of distilled water (adjusted to pH1 by the addition of 0.001 M hydrochloric acid) and 8.5 ml of methanol (giving a final methanol concentration of 550/,). The final mixture was applied to the Cl8 cartridge at a flow rate of 2 ml/min, as were all subsequent washes and elution. When the application was complete, one of two
Table 3. Effect of increasing volumes of plasma on the recovery of added tritiated 25-OHD, applying methyl cyanide extracts to Sep-Pak Cl8 cartridges Volume of plasma extracted (ml)
‘? Recovery* Without (NH,),SO,
With (NH&SO,
1 2 5 10 I5 20
88.5 89.5 92.9 75.4 53.7 40.6
84.0 76.0 83.4 90.1 82.3 80.2
*Values recorded are the means of duplicate experiments. Details of the extraction and elution procedures with and without ammonium sulphate are given in the “Method” section in the text.
(‘. .1.
RH0I)I.S(‘I td.
Table 4. Rccoverles of added tritiated secosteroids from plasma (3 ml) applied to Sep-Pak C I8 cartridges using the procedure of Dabek (J, rr1.[7]. with and without subsequent fractionation of Sep.Pak CIX and SIL cartridges
Secohteroid D; 25.OHD, 24,25(OH),D,
1.25(OH),D,
“(, Recovery (mean i SD. ~1: 5) Fractionated* Fractionated: Total* on Cl8 on SII. 66.7 * 3.3 93.4 + 4.0 97.3 f 4.8 98. I f 0.8
Xl.7 + 10.0
63. I * 3.I x9.2 * 3.x 91.1 i4.5 89.7 * 1.5
*Details of these two procedures are given in the text. The use of silanised Sep-Pak Cl8 cartridges had no effect on the recovery of any of the secosteroids studied. tThese fractions were obtained by sequentially eluting Sep-Pak SIL cartridges with isopropanol (isoP) and light petroleum (80-lW)AR (LP) mixtures at 4 C. Fraction I: 0-3ml. isoP LP( I : 9. v’v-discard; Fraction 2: 3-l 5 ml, isoP-LP( I 9. v:\)contams D: Fraction 3: 15-25 ml. isoP-LP(3 : 97. v, v) contains 25.OHD: Fraction 4: 25-45 ml. isoP-LP( I : 9. v/v)---contains 24.25. (OH),D ‘:md 1,25(OH),D and recovery Values were thus obtained separately.
alternative procedures was carried out: (a) Total elution: The cartridge was washed with 3 ml methanol-Hz0 (7:3, v/v) and the secosteroids were eluted with 4ml methyl cyanide. (b) Fractionation: Dabek et n1.[7] described a procedure for the separation of the secosteroids into three fractions containing: vitamin D, 25-OHD and the polyhydroxylated metabolites of vitamin D. In this paper only the 25-OHD fraction was examined. Table 4 gives the recoveries obtained using these procedures. Each extract was examined by HPLC and GC-MS and the amount of material extracted weighed. The weight of the extracts obtained from 2 ml plasma using the method of Dabek et a/.[71 (Total: 21.4 mg, fractionated 25-OHD: 6.2 mg) were considerably higher than
that obtained using the methyl cyanide extraction procedure: 0.8 mg). It must be emphasised that if plasma samples which have been stored at - 20°C are used in the Dabek procedure, it is very important to centrifuge before use. If this is not done, particulate material may make it impossible to pass the extracts through the Sep-Pak cartridges. Silanisation of the Sep-Pak cartridges prior to use had no effect on the recoveries of any of the secosteroids studied using the procedure of Dabek et rr/.[7]. It was found that none of the extracts were suitable for direct examination by GC-MS or by HPLC. Figure 2 gives comparative HPLC traces on Zorbax Sil comparing 2%OHD extracts prepared by the methyl cyanide extraction procedure and by the fractionation procedure of
Fig. I. Straight-phase HPLC of 25.OHD extracts from Sep-Pak Cl8 cartridges. Plasma (3 ml) was extracted by (A) the methyl cyanide procedure and (B) the fractionated Dabek procedure [7]. Zorbax-SIL (IOpm, 250 x 0.47 mm) columns were eluted with hexane-isopropanol (95:5,v/v) at a flow rate of I .5 ml/min. Eluent was monitored at 264 nm (A) O.OIAUFS, (B) 0. IAUFS. The position of 25-OHD, is indicated by an arrow. The recovery of ‘H 25-OHD, was 66.0”,:, after HPLC of the methyl cyamde extract (A) and the plasma concentration of 25-OHD, in this extract was 7.3 ng:ml.
Vitamin D, and metabolite extraction by Sep-Pak Dabek ef a1.[7]. In order to obtain a trace from the Dabek extract the amplification was reduced to ten times Iess than that used for the trace from the methyl cyanide extract and thus the 25-OHD cannot be seen in the Dabek extract. However, if the Dabek extract eluted from the C 18 cartridge was subsequently fractionated on Sep-Pak SIL cartridge, fractions suitable for direct HPLC were obtained and overall recoveries (with the exception of that for vitamin D) were very similar to those obtained from methyl cyanide extracts eluted directly from Ct8 cartridges. lise of’ Sep-Pnk
SIL for fractionation
of vitamin D
metabolites
Adams et al.]1 l] described a fractionation system using Sep-Pak SIL cartridges which ahowed for the separation of vitamin D, 2%OHD, 24,25(OH),R and 1,25(OH),D. Experiments showed that by changing the solvent system described by Adams et a/.[ 1l] and using isopropanol-light petroleum mixtures for elution at 4°C improved resolution between, and quantitative recoveries of, all four secosteroids could be obtained. Recoveries of added secosteroids extracted from plasma by the method of Dabekfq, eluted and fractionated on Sep-Pak SIL cartridges as described above are given in Table 4.
Sep-Pak Cl8 reverse phase cartridges have been successfully used for the extraction of steroid hormone metabolites from urine [5] and plasma [6], estrogens from urine and plasma [lo]. These reverse phase cartridges have also been used in methods for the estimation of pfasma concentrations of 2%OHQ and and 25-OHD, [7,8, 121, 1,25(OH),D, 1,25(OH),D? [12, 131. The majority of these procedures use methyl cyanide extraction prior to application to the Sep-Pak cartridge. No comprehensive study of the use of methyl cyanide and Sep-Fak Cl8 cartridges for the extraction of vitamin D and its metabolites from urine and plasma has hitherto been published. There are two methods of applying plasma to Sep-Pak cartridges. The method of Dabek et a/.[71 utilises acidified methanolic plasma, while the alternative method examined in detail here removes protein by precipitation with methyl cyanide and centrifugation, and the aqueous methyl cyanide supernatant is applied to the Sep-Pak. Superficially the method of Dabek et a1.[7] appears to be advantageous, since it does not require a centrifugation step to remove precipitated protein and gives higher recoveries than the methyt cyanide method. Centrifugation to remove fibrin from stored plasma is, however, still necessary. Methyl cyanide extracts have been cleaner than those obtained using the Dabek method and have proved to be suitable for direct HPLC analysis[8]. Extracts prepared using the
1353
method of Dabek[7] have had to be further fractionated using SegPak SIL cartridges before they are suitable for HPLC. unfortunately vitamin D is difficult to extract using the methyl cyanide-Sep-Pak procedure described. Only one third of tritiated D, added to plasma can be extracted into the methyl cyanide layer and only one eighth of that is adsorbed onto the material of the cartridge. 11, is substantially more hydrophobic than its metabol~tes and is thus less easily extracted by a potar solvent such as methyl cyanide. The Dabek procedure[7] atso does not appear to give quantitative recoveries of D, although it gives recoveries (66.7%) which are roughly twice the mean obtained by methyl cyanide (26.6%). Silanisation of the Sep-Pak cartridge allows the adsorption of all the D, in the methyl cyanide layer, implying that the presence of polar groups in untreated Sep-Pak cartridges prevents the complete adsorption of D,. Silanisation has no effect on the adsorption of any of the metabolites nor does it increase the extraction of D, by the Dabek procedure. interestingly enough cholesterol behaves in a similar fashion to vitamin D and only 5% of free cholesterol is found in the methyl cyanide layer after extraction of plasma with methyl cyanide. The majority of D, which remains in the protein pellet can be recovered by dispersion and extraction with methanold~chloromethane (3. f, v/v) leading to an overall recovery of SOY/,. While Sep-Pak Cl8 cartridges offer a rapid and simple method for the extraction of the metabolites of vitamin D neither the methyl cyanide extraction nor the method of Dabek et a1.[7] gives satisfactory extraction of vitamin D itself. Sep-Pak cartridges can be used for a relatively crude separation of vitamin D metabolites [7] but our experience would suggest that the separation achieved on these reverse-phase cartridges is inadequate and improved results can be achieved by subsequent fractionation on straightphase Sep-Pak ]I l]. ~ckno~~ed~eme~ts-~~s work has been supported by grants from the Wellcome Trust, The Medical Research Council and the Research Advisory Committee of the London Hospital, for which we arc extremely grateful REFERISNXXS
Al-Dujaili E. A. S. and Edwards C. R. W.: Development and application of a direct radioimmunoassay for plasma aldosterone using I-125 labelled linandcomparison of three method. 7. clin. Endocr. Me&b. 46, (1978) 105-I 13.
Schonesbofer M., Fenner A. and Dulce H. J.: Interferences in the radio~mmunolo~cai det~~~nation of urinary free eortisol. C&n. e&z. Arta 101 (1980) 125-l 34. 3. Haddad J. G.: Vitamin D metabolites. In Methods qf Hormone Radioimmunoassay (Edited by J. M. Jaffe and H. R. Behrman). Academic Press. New York. San Francisco London (1979) pp. 437-452. 4. Taylor G. A., Peacock M., Pelt B., Brown W. and Holmes A.: Purification of plasma vitamin D metabo-
lites for radioimmunoassay. Clin. chin?. AC/U 108 (1980) 239246. Shackleton C. 2-I. L. and Whitney J. 0.: Use of Sep-Pak cartridges for urinary steroid extraction: evaluation of the method for use prior to gas chromatographic analysis. Clirr. r&r. AC& 107 (1970) 231-243. Cannel1 G. R., Galligan J. P., Mortimer R. H. and Thomas M. J.: Extraction of steroids from plasma with reverse phase Cl8 cartridges. C/in. c/Cm. AL.IU 122 (1982) 419-423. Ddbek J. T., Harkonen M.. Wahlroos 0.. Adlercreutz H.: Assay for plasma 25-hydroxyvitam~n D2 and 25.h~droxyvitamin D, by Lehigh-perfo~an~” liquid chromatography. C/in. C&m. 27 (1981) 13461351. 8. Turnbull H., Trafford D. J. H. and Makin H. L. J.: A rapid and simple method for the measurement of plasma 25-hydroxyvitamin D, and 25-hydroxyvitamin D, using Sep-Pak Cl8 cartridges and a single highperformance liquid chromatographic step. Clin. chim. Arra 120 (1982) 65-76.
9 Seamark D. A., Tratl’ord II. J. t-1 fItscock\ P. G. and Makin H. L. .I.: Iligh-perform~tnce liquid chromatography of vitamin I>: enhanced ultraviolet absorbance by prior conversion to isotochysterol dcrivatitcs. J. C~~~~~~~~~~J~. 197 (1980)271 273. H.: Reversed10 Heikkinen R., Fotsis T. and Adlcrcreutz phase C,, cartridges for extraction of estrogens from urine and plasma: Clin. Chem. 27 (1981) II86 11x9. I I Adams J. S., Clemens T L. and Holick M. t:.: Silica Scp-Pak preparative chromatographv for vttamin D and its metabolites. J. f.hromrrl& i& ( 19XI ) 19X--20 I_ 12 Fraher L. J., Jones G.. Clemens T. L.. Adami S. and O’Riordan J. L. H.: Radio-assays of vitamin DI and D; mctabohtes. Advance abstracts ofpapcrs for the Xlllth Acta Endocrinologica Congress. Cambridge, August 198 I. Abstract No. X. .~
0022-4731/83 S3.00+0.00 Copyright(c;1983 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
AN EVALUATION OF THE USE OF SEP-PAKt Cl8 CARTRIDGES FOR THE EXTRACTION OF VITAMIN D, AND SOME OF ITS METABOLITES FROM PLASMA AND URINE C. J. RHODES, P. A. CLARIDGE, D. J. H. TRAFFORD and Steroid
Laboratory,
Department
of Chemical Pathology, The London Street, London El 2AD, UK
H. L. J. MAKIN*
Hospital
Medical
College,
Turner
(Received 15 November 1982) Cl8 cartridges for the extraction of vitamin D and some of its metabolites by studying the recovery of added tritiated secosteroids. The preparation of the cartridges, recoveries, extraction and elution with a number of solvents, effect of varying flow rates for application and elution, and the effect of increasing volumes of plasma and urine have been investigated. Two methods for the application of secosteroids present in plasma to Sep-Pak Cl8 cartridges have been examined, using methyl cyanide extracts removing precipitated protein by centrifugation, and using acidified methanolic plasma. Methyl cyanide extracts applied to Sep-Pak Cl8 cartridges and eluted with methanol or methyl cyanide gave the cleanest extracts suitable for direct HPLC. Acidified methanolic plasma, applied to Sep-Pak Cl8 cartridges and eluted with methanol or methyl cyanide gave extracts which could not be applied directly to an HPLC-further fractionation using Sep-Pak XL cartridges was necessary. Recoveries of added tritiated secosteroids using both methods were greater than 80% with the exception of vitamin D itself which was poorly recovered-methyl cyanide extraction giving only 30% recovery and use of acidified methanolic plasma giving 66% recovery.
Summary-The
use of Sep-Pak
from plasma and urine has been evaluated
INTRODUCTION
Although radioimmunoassays for some steroid hormones have been described which can be carried out directly on plasma [I] or urine, some criticisms have been levelled at their specificity [2]. Unlike such steroids, a prerequisite for the successful analysis of vitamin Dt and its metabolites in plasma or urine by any procedure, is, at the moment, their extraction from the aqueous medium pri2r to chromatography and/or quantitation. Quantitative extraction with organic solvents can be achieved and many assays use time consuming, multi-extraction procedures to obtain reasonable recoveries [3] using large volumes of organic solvents which have to be evaporated. A study of four different solvent extraction procedures prior to radioimmunoassay has been described [4]. This paper describes the evaluation of an alternative approach, using reverse-phase Sep-Pak Cl8 cartridges for the preliminary removal of vitamin D and some of its metabolites from plasma and urine. *To whom correspondence should be sent. TRegistered Trade Mark. fsystematic and trivial names of vitamin D and its derivatives used in this paper are: Vitamin D, (9,10-seco5,7,10(19),22-ergostatetraen-38-01): D,. Vitamin D, (9,10-seco-5,7,10(19)-cholestatrien-3~-o~): D,. 25-Hyhroxyvitamin D,: 25-OHD,. 25-Hydroxyvitamin D,: 25-OHD,. 24.25-dihvdroxvvitamin D,: 24,25(OH),D,. lcc,25-diiydroxyvitamin D;: 1,25(0H)&. The term b is used when it is not required to distinguish between Dz and D,.
Although Sep-Pak Cl8 cartridges have been used for the extraction of steroid hormone metabolites from urine [5] and plasma [6] and for the extraction [7, 81 and fractionation [7] of 25-hydroxyvitamin D from plasma prior to high-performance liquid chromatography (HPLC), no proper evaluation of their use for the extraction of vitamin D and its metabolites has previously been published. MATERIALS AND METHODS Reagents
25 Ci/mmol), [ 1,2-‘HIvitamin D, act. (SP. act.: 25_hydroxy[23,24-‘HIvitamin D, (sp. 102 Ci/mmol), 24,25-dihydroxy[23,24-3H]vitamin D, act.: 77 Ci/mmol) and Ir,25-dihydroxy(sp. [23,24-3H]vitamin D3 (sp. act.: 120 Ci/mmol) were all obtained from Amersham International PLC. (Amersham, Bucks., UK) and were found to be radiochemically pure ( > 98%) on receipt but were re-purified immediately prior to use by highperformance liquid chromatography (HPLC). When added to plasma or urine, irrespective of volume, for recovery experiments, 20,000 dpm of each secosteroid was used unless otherwise stated. Other reagents used, unless otherwise specified, were the highest grade available (usually Analytical Reagent) from BDH Chemicals Ltd (Poole, Dorset, UK) and were used as supplied without further purification. Solvents used for HPLC were obtained from Rathburn Chemicals Ltd. (Peebleshire, UK) and HPLC was
I349
carried out as described previously [8,9]. HPLC solvents were degassed before use. Sep-Pak Cl8 and SIL cartridges were purchased from Waters Associates (UK) Ltd (Northwich. Cheshire, UK). Liquid scintillation counting was carried out using an fntertechnique model SL3000 (Kontron Intertechnique Ltd. St. Albans, Herts., UK) and 5 ml of NE250 liquid scintillation counting fluid. At least 1000 counts were collected. Unquenched tritium efficiency was around SW!<,falling to around 40”,/, for the most severely quenched samples, and the background count rate was approx 30cpm. Quench correction, where necessary, was carried out using an external added standard (jH hexadecane, Amersham International Ltd).
Sep-Pak Cl8 cartridges were prepared before use by successively washing each cartridge with 10 ml of methanol and 10ml of water. Radioactive secosteroids were added to aliquots of urine or plasma which, after incubation at 37°C for 15 min and room temperature for 45 min, were extracted by shaking on a vortex mixer for 60s with an equal volume of methyl cyanide. In the case of plasma, the precipitated protein was removed by centrifugation (15OOg for 10 min), and the extract was then applied rapidly (flow rate > 24 ml/min) to a Sep-Pak cartridge. The cartridge was then with 3ml of washed methanol-water (70:30, v/v) and the vitamin D metabolites eluted by slowly (flow rate < 24 ml/min) passing through the cartridge, 4 ml of methyl cyanide. In cases where the volume of plasma or urine was greater than 5 ml, 0.5 g of ammonium sulphate per ml of original sample was added to the methyl cyanide extract before it was applied to the Sep. Pak Cl8 cartridge. The mixture was shaken and centrifuged (I 500g for 10 min). The clear upper methyl cyanide layer was removed, evaporated to dryness under a gentle stream of nitrogen at 37”C, taken up again in 10 m! of methyl cyanide-water (I : 1 v/v) and then applied to the Sep-Pak cartridge, which was washed Table I. Recoveries of added tritiated secosteroids from plasma (2 ml) and urine (2 ml), applying methyl cyanide extracts to Sep-Pak Cl8 cartridges Secosteroid D, 25OHD, 24,25(OH)zDT 1.25(OH),D,
“i, Recovery (mean f SD, n = 5) Urine Plasma 73.5 + 10.9 91.7 $- 3.2 79.9 * 5.2 88.6 * 7.1
6.9 i 84.9 + 83.9 * 82.6 k
3.3* 9.8 14.0 3.9
*Using silanised Sep-Pak cartridges, this recovery can be increased to give a quantitative recovery, after dUtiOn from the Sep-Pak cartridge, of the total amount extracted into the methyl cyanide layer (26.6 + 7.10/,). Use of silanised Sep-Pak cartridges does not affect the recoveries of other secosteroids examined. Recoveries of all secosteroids, other than D, in the methyl cyanide extract was quantitative (Plasma 102.4 k 5.3, n = 15; urine 101.6 f 9.7, n = L5). Details of extraction and elution procedures are given in the “Method” section in the text.
with methanol-water as descibed above.
and eluted w~tb methyl c’!;mi _
RESU
f.‘l’S
Preparation c!f Sep -Pa.4 curtridgw Sep-Pak Cl 8 cartridges were prepared before use as recommended by the manufacturers by passing methanol and water through the cartridge ;ts dcscribed in the Method section above. Experiments using tritiated 2S-OHD,, 24.25(OH),D,. and 1,25(OH),D, showed that the length of time between preparation and use was not crucial since similar recoveries of added tritiated secosteroids were ohtained using cartridges prepared immediately and up to 4 h before use.
Recovery studies Tritiated secosteroids were added to plasma and urine which were processed as described in the Method section above. The final extract was counted on a scintillation counter to assess recovery. HPLC was carried out on some of the extracts and in no case was the radioactivity not eluted from the HPLC with the appropriate standard secosteroid. Table I gives the results of these experiments. In an attempt to increase the recoveries of added secosteroids. SepPak C 18 cartridges were “silanised” by soaking in I ‘I(, dimethyldichlorisilane in toluene for I h. washed with methanol and finally prepared as described above. Silanisation of Sep-Pak Cl8 cartridges had no ctfect on the recovery of any of the secosteroids studied except for vitamin D,. the recovery of which, in this experiment, was increased from a mean of 6.7”,, to a mean of 3 I .7’!$
Extraction md elthrm studies Tritiated secosteroids were added to 2 ml aliyuots of plasma and extracted with a variety of solvents (methanol, ethanol, acetone, methyl cyanide). Results were similar to those reported previously [8]-methyl cyanide consistently gave the highest recovery of all the steroids examined also giving the cleanest extracts as judged by HPLC analysis. There seemed to be little difference, however, between methanol and methyl cyanide as eluting solvents: both giving clean extracts and similar recoveries. All tritiated secosteroids examined were eluted completely from the cartridge with 4mI of either methanol or methyl cyanide. In an attempt to fractionate the secosteroids, methyl cyanide extracts of plasma to which tritiated secosteroids had been added were applied to cartridges which were then etuted with methanol- water mixtures. Poor elution profiles, similar to those reported previously [7], were obtained with inadequate resolution between the secosteroids.
Effect of vwying ,fh
rutrs of’ upplicutiorz and drtfim
Considerable variation in recovery was initially experienced, so the speed of application of methyl
1351
Vitamin D, and metabolite extraction by Sep-Pak Table 2. Effect of varying flow rates of application to and elution from Sep-Pak Cl8 cartridges using the methyl cyanide extraction method Flow rate (ml/min) 144
24 19 12 2
% Recovery of added ‘H 25-OHD, Application* Elution 83.6 89.9 54.3 50.0 36.5
49.5 84.8 80.0 85.6 81.2
*Methyl cyanide extracts were applied at the different flow rates indicated (and washed and eluted at a flow rate of 7 ml/min) or applied at 30ml/min and washed and eluted at the flow rates indicated. Values recorded are the means of duplicate experiments. Details of elution and extraction are given in the “Method” section.
cyanide extracts to the Sep-Pak cartridge and the subsequent rate of elution was examined. Cartridges were attached to a peristaltic pump (AA11 AutoAnalyser), which could pump solvent through at different flow rates (2-144 ml/min). The recovery of tritiated 25-OHD, obtained when methyl cyanide extracts of plasma were applied to and eluted from the Sep-Pak cartridge at different flow rates were measured. The highest recoveries were obtained when extracts were applied rapidly (24144 ml/min) and were eluted slowly ( < 24 ml/min). Details of recoveries at different flow rates are given in Table 2. Effect of increasing volumes of plasma and urine Large volumes (> 5 ml) of plasma and urine, extracted with the appropriate volume of methyl cyanide gave rise to low recoveries of added tritiated 25-OHD, from the Sep-Pak cartridge. Evaporating the methyl cyanide extract to dryness, which is a lengthy procedure because of the large amount of water present, and subsequent application of the dried extract, re-dissolved in 10 ml methylcyanide: water (1: 1, v/v) to the cartridge did partially improve recovery but only to around 80% of that achieved with small volumes of plasma ( < 5 ml). If ammonium sulphate (0.5g per ml of sample) was added to the methyl cyanide extract before application to the Sep-Pak Cl8 cartridge, shaken and then centrifuged, two immiscible layers were obtained. The clear upper layer contained methyl cyanide which was free of water and could thus be more easily evaporated. If this dried extract was re-dissolved in 10 ml methyl cyanide-water (1: 1, v/v) and re-applied to the cartridge, recoveries were completely restored to that obtained using small volumes of plasma and urine. Table 3 shows the effect on the recovery of added ‘H 25-OHD,, of extracting increasing volumes of plasma and applying the extract to Sep-Pak cartridges before and after treatment with ammonium sulphate. Steroid hormone metabolites have been recovered from urine by passing it directly through Sep-Pak Cl8 cartridges[5] and eluting the adsorbed steroids
with methanol. Increasing volumes of urine (5-lOOml), to which ‘H 25-OHD, had been added, were passed through Sep-Pak cartridges, which were then washed with methanol-water and eluted with methyl cyanide as described in the Methods section above. As the volume of urine used increased the recovery of tritiated 25-OHD, dropped (from 91.7% using 2 ml to 14.6% using 100 ml). However, if the methyl cyanide extract of urine was treated with ammonium sulphate as described for plasma in the preceeding paragraph, added tritiated 25-OHD, could be recovered from large volumes of urine (20ml of urine applied directly gave a recovery of 34.9% but when extracted, and treated with ammonium sulphate, recovery was restored to that achieved using small volumes of urine). Examination tridges
of other procedures using Sep -Pak car -
Other procedures for the extraction of 25-OHD [7], oestrogens [lo] and steroids [6] from plasma using reverse-phase Sep-Pak cartridges have been described. These procedures do not use methyl cyanide extraction but instead directly apply to the Sep-Pak column acidified plasma [6, lo] or acidified methanolic plasma [7]. All three procedures were studied using tritiated 25-OHD, added to 5 ml of plasma. Only the method of Dabek et af[7] using acidified methanolic plasma gave reasonable recoveries, the other two procedures, using acidified plasma without methanol, gave recoveries of less than 10% of added 25-OHD,. The method of Dabek et al.[7] was therefore studied using all four tritiated secosteroids. Each secosteroid was added to 3 ml aliquots of plasma and after incubation, the plasma was treated as described by Dabek et a1.[7]. This procedure involves acidifing the plasma by addition of 4ml of distilled water (adjusted to pH1 by the addition of 0.001 M hydrochloric acid) and 8.5 ml of methanol (giving a final methanol concentration of 550/,). The final mixture was applied to the Cl8 cartridge at a flow rate of 2 ml/min, as were all subsequent washes and elution. When the application was complete, one of two
Table 3. Effect of increasing volumes of plasma on the recovery of added tritiated 25-OHD, applying methyl cyanide extracts to Sep-Pak Cl8 cartridges Volume of plasma extracted (ml)
‘? Recovery* Without (NH,),SO,
With (NH&SO,
1 2 5 10 I5 20
88.5 89.5 92.9 75.4 53.7 40.6
84.0 76.0 83.4 90.1 82.3 80.2
*Values recorded are the means of duplicate experiments. Details of the extraction and elution procedures with and without ammonium sulphate are given in the “Method” section in the text.
(‘. .1.
RH0I)I.S(‘I td.
Table 4. Rccoverles of added tritiated secosteroids from plasma (3 ml) applied to Sep-Pak C I8 cartridges using the procedure of Dabek (J, rr1.[7]. with and without subsequent fractionation of Sep.Pak CIX and SIL cartridges
Secohteroid D; 25.OHD, 24,25(OH),D,
1.25(OH),D,
“(, Recovery (mean i SD. ~1: 5) Fractionated* Fractionated: Total* on Cl8 on SII. 66.7 * 3.3 93.4 + 4.0 97.3 f 4.8 98. I f 0.8
Xl.7 + 10.0
63. I * 3.I x9.2 * 3.x 91.1 i4.5 89.7 * 1.5
*Details of these two procedures are given in the text. The use of silanised Sep-Pak Cl8 cartridges had no effect on the recovery of any of the secosteroids studied. tThese fractions were obtained by sequentially eluting Sep-Pak SIL cartridges with isopropanol (isoP) and light petroleum (80-lW)AR (LP) mixtures at 4 C. Fraction I: 0-3ml. isoP LP( I : 9. v’v-discard; Fraction 2: 3-l 5 ml, isoP-LP( I 9. v:\)contams D: Fraction 3: 15-25 ml. isoP-LP(3 : 97. v, v) contains 25.OHD: Fraction 4: 25-45 ml. isoP-LP( I : 9. v/v)---contains 24.25. (OH),D ‘:md 1,25(OH),D and recovery Values were thus obtained separately.
alternative procedures was carried out: (a) Total elution: The cartridge was washed with 3 ml methanol-Hz0 (7:3, v/v) and the secosteroids were eluted with 4ml methyl cyanide. (b) Fractionation: Dabek et n1.[7] described a procedure for the separation of the secosteroids into three fractions containing: vitamin D, 25-OHD and the polyhydroxylated metabolites of vitamin D. In this paper only the 25-OHD fraction was examined. Table 4 gives the recoveries obtained using these procedures. Each extract was examined by HPLC and GC-MS and the amount of material extracted weighed. The weight of the extracts obtained from 2 ml plasma using the method of Dabek et a/.[71 (Total: 21.4 mg, fractionated 25-OHD: 6.2 mg) were considerably higher than
that obtained using the methyl cyanide extraction procedure: 0.8 mg). It must be emphasised that if plasma samples which have been stored at - 20°C are used in the Dabek procedure, it is very important to centrifuge before use. If this is not done, particulate material may make it impossible to pass the extracts through the Sep-Pak cartridges. Silanisation of the Sep-Pak cartridges prior to use had no effect on the recoveries of any of the secosteroids studied using the procedure of Dabek et rr/.[7]. It was found that none of the extracts were suitable for direct examination by GC-MS or by HPLC. Figure 2 gives comparative HPLC traces on Zorbax Sil comparing 2%OHD extracts prepared by the methyl cyanide extraction procedure and by the fractionation procedure of
Fig. I. Straight-phase HPLC of 25.OHD extracts from Sep-Pak Cl8 cartridges. Plasma (3 ml) was extracted by (A) the methyl cyanide procedure and (B) the fractionated Dabek procedure [7]. Zorbax-SIL (IOpm, 250 x 0.47 mm) columns were eluted with hexane-isopropanol (95:5,v/v) at a flow rate of I .5 ml/min. Eluent was monitored at 264 nm (A) O.OIAUFS, (B) 0. IAUFS. The position of 25-OHD, is indicated by an arrow. The recovery of ‘H 25-OHD, was 66.0”,:, after HPLC of the methyl cyamde extract (A) and the plasma concentration of 25-OHD, in this extract was 7.3 ng:ml.
Vitamin D, and metabolite extraction by Sep-Pak Dabek ef a1.[7]. In order to obtain a trace from the Dabek extract the amplification was reduced to ten times Iess than that used for the trace from the methyl cyanide extract and thus the 25-OHD cannot be seen in the Dabek extract. However, if the Dabek extract eluted from the C 18 cartridge was subsequently fractionated on Sep-Pak SIL cartridge, fractions suitable for direct HPLC were obtained and overall recoveries (with the exception of that for vitamin D) were very similar to those obtained from methyl cyanide extracts eluted directly from Ct8 cartridges. lise of’ Sep-Pnk
SIL for fractionation
of vitamin D
metabolites
Adams et al.]1 l] described a fractionation system using Sep-Pak SIL cartridges which ahowed for the separation of vitamin D, 2%OHD, 24,25(OH),R and 1,25(OH),D. Experiments showed that by changing the solvent system described by Adams et a/.[ 1l] and using isopropanol-light petroleum mixtures for elution at 4°C improved resolution between, and quantitative recoveries of, all four secosteroids could be obtained. Recoveries of added secosteroids extracted from plasma by the method of Dabekfq, eluted and fractionated on Sep-Pak SIL cartridges as described above are given in Table 4.
Sep-Pak Cl8 reverse phase cartridges have been successfully used for the extraction of steroid hormone metabolites from urine [5] and plasma [6], estrogens from urine and plasma [lo]. These reverse phase cartridges have also been used in methods for the estimation of pfasma concentrations of 2%OHQ and and 25-OHD, [7,8, 121, 1,25(OH),D, 1,25(OH),D? [12, 131. The majority of these procedures use methyl cyanide extraction prior to application to the Sep-Pak cartridge. No comprehensive study of the use of methyl cyanide and Sep-Fak Cl8 cartridges for the extraction of vitamin D and its metabolites from urine and plasma has hitherto been published. There are two methods of applying plasma to Sep-Pak cartridges. The method of Dabek et a/.[71 utilises acidified methanolic plasma, while the alternative method examined in detail here removes protein by precipitation with methyl cyanide and centrifugation, and the aqueous methyl cyanide supernatant is applied to the Sep-Pak. Superficially the method of Dabek et a1.[7] appears to be advantageous, since it does not require a centrifugation step to remove precipitated protein and gives higher recoveries than the methyt cyanide method. Centrifugation to remove fibrin from stored plasma is, however, still necessary. Methyl cyanide extracts have been cleaner than those obtained using the Dabek method and have proved to be suitable for direct HPLC analysis[8]. Extracts prepared using the
1353
method of Dabek[7] have had to be further fractionated using SegPak SIL cartridges before they are suitable for HPLC. unfortunately vitamin D is difficult to extract using the methyl cyanide-Sep-Pak procedure described. Only one third of tritiated D, added to plasma can be extracted into the methyl cyanide layer and only one eighth of that is adsorbed onto the material of the cartridge. 11, is substantially more hydrophobic than its metabol~tes and is thus less easily extracted by a potar solvent such as methyl cyanide. The Dabek procedure[7] atso does not appear to give quantitative recoveries of D, although it gives recoveries (66.7%) which are roughly twice the mean obtained by methyl cyanide (26.6%). Silanisation of the Sep-Pak cartridge allows the adsorption of all the D, in the methyl cyanide layer, implying that the presence of polar groups in untreated Sep-Pak cartridges prevents the complete adsorption of D,. Silanisation has no effect on the adsorption of any of the metabolites nor does it increase the extraction of D, by the Dabek procedure. interestingly enough cholesterol behaves in a similar fashion to vitamin D and only 5% of free cholesterol is found in the methyl cyanide layer after extraction of plasma with methyl cyanide. The majority of D, which remains in the protein pellet can be recovered by dispersion and extraction with methanold~chloromethane (3. f, v/v) leading to an overall recovery of SOY/,. While Sep-Pak Cl8 cartridges offer a rapid and simple method for the extraction of the metabolites of vitamin D neither the methyl cyanide extraction nor the method of Dabek et a1.[7] gives satisfactory extraction of vitamin D itself. Sep-Pak cartridges can be used for a relatively crude separation of vitamin D metabolites [7] but our experience would suggest that the separation achieved on these reverse-phase cartridges is inadequate and improved results can be achieved by subsequent fractionation on straightphase Sep-Pak ]I l]. ~ckno~~ed~eme~ts-~~s work has been supported by grants from the Wellcome Trust, The Medical Research Council and the Research Advisory Committee of the London Hospital, for which we arc extremely grateful REFERISNXXS
Al-Dujaili E. A. S. and Edwards C. R. W.: Development and application of a direct radioimmunoassay for plasma aldosterone using I-125 labelled linandcomparison of three method. 7. clin. Endocr. Me&b. 46, (1978) 105-I 13.
Schonesbofer M., Fenner A. and Dulce H. J.: Interferences in the radio~mmunolo~cai det~~~nation of urinary free eortisol. C&n. e&z. Arta 101 (1980) 125-l 34. 3. Haddad J. G.: Vitamin D metabolites. In Methods qf Hormone Radioimmunoassay (Edited by J. M. Jaffe and H. R. Behrman). Academic Press. New York. San Francisco London (1979) pp. 437-452. 4. Taylor G. A., Peacock M., Pelt B., Brown W. and Holmes A.: Purification of plasma vitamin D metabo-
lites for radioimmunoassay. Clin. chin?. AC/U 108 (1980) 239246. Shackleton C. 2-I. L. and Whitney J. 0.: Use of Sep-Pak cartridges for urinary steroid extraction: evaluation of the method for use prior to gas chromatographic analysis. Clirr. r&r. AC& 107 (1970) 231-243. Cannel1 G. R., Galligan J. P., Mortimer R. H. and Thomas M. J.: Extraction of steroids from plasma with reverse phase Cl8 cartridges. C/in. c/Cm. AL.IU 122 (1982) 419-423. Ddbek J. T., Harkonen M.. Wahlroos 0.. Adlercreutz H.: Assay for plasma 25-hydroxyvitam~n D2 and 25.h~droxyvitamin D, by Lehigh-perfo~an~” liquid chromatography. C/in. C&m. 27 (1981) 13461351. 8. Turnbull H., Trafford D. J. H. and Makin H. L. J.: A rapid and simple method for the measurement of plasma 25-hydroxyvitamin D, and 25-hydroxyvitamin D, using Sep-Pak Cl8 cartridges and a single highperformance liquid chromatographic step. Clin. chim. Arra 120 (1982) 65-76.
9 Seamark D. A., Tratl’ord II. J. t-1 fItscock\ P. G. and Makin H. L. .I.: Iligh-perform~tnce liquid chromatography of vitamin I>: enhanced ultraviolet absorbance by prior conversion to isotochysterol dcrivatitcs. J. C~~~~~~~~~~J~. 197 (1980)271 273. H.: Reversed10 Heikkinen R., Fotsis T. and Adlcrcreutz phase C,, cartridges for extraction of estrogens from urine and plasma: Clin. Chem. 27 (1981) II86 11x9. I I Adams J. S., Clemens T L. and Holick M. t:.: Silica Scp-Pak preparative chromatographv for vttamin D and its metabolites. J. f.hromrrl& i& ( 19XI ) 19X--20 I_ 12 Fraher L. J., Jones G.. Clemens T. L.. Adami S. and O’Riordan J. L. H.: Radio-assays of vitamin DI and D; mctabohtes. Advance abstracts ofpapcrs for the Xlllth Acta Endocrinologica Congress. Cambridge, August 198 I. Abstract No. X. .~