Determination of prednisolone,prednisone, and cortisol in human plasma by high-performance liquid chromatography

A method is described for the measurement of prednisone and prednisolone in human plasma by high-performance liquid chromatography (hplc). An ether/dichloromethane extract (60:40, $\text{v}\text{v}$) of plasma is evaporated to dryness and chromatographed on a 250 × 4.5-mm Whatman Partisil column with uv detection at 239 nm. Prednisone, prednisolone cortisol, and the internal standard dexamethasone are satisfactorily resolved with the elution system of water-saturated dichloromethane/ethanol (95:4, $\text{v}\text{v}$). The hplc method can be used for plasma prednisolone concentrations as low as 25 ng/ml. The values correlate well with those obtained by a radioimmunoassay procedure for prednisolone.     

Prednisolone and prednisone neo-formation in bovine urine after sampling

The rise in the frequency of detecting prednisolone in bovine urine from northern Italy has come into focus of attention in recent years. The possibility that neo-formation of prednisolone or that prednisone may occur in urine after collection of samples was therefore investigated. Cow urine collected for official routine controls in Lombardy containing more than 80 ng/ml cortisol, and prednisolone and prednisone below the decision limit (CCα) of the method (0.4 and 0.5 ng/ml, respectively) was used. The C1-2 dehydrogenation of naturally present cortisol and cortisone was checked by incubating urine, both contaminated and uncontaminated with faeces, at 37°C and by collecting samples at 0, 1, 2, 4, 6 and 24 h. The influence of Helix pomatia juice was also investigated in order to determine whether deconjugation could influence the reliability of the results. All samples were analysed by HPLC-MS3 for the presence of cortisol, cortisone, prednisolone and prednisone in negative electrospray ionisation mode, utilising the consecutive reaction monitoring of product ions derived from the formate molecular adduct ([M+HCOO]-). The observed neo-formation of prednisolone shows that inappropriate temperatures in sample storage and processing can result in an incorrect accusation of non-compliance. The faecal contamination of urine, performed with the aim to mimic a collection conducted without the necessary care, moreover, evoked a high increase in prednisolone concentration in two out of seven animals. Moreover, H. pomatia juice had no significant effect on the prednisolone concentration, indicating that this corticosteroid is present in its free form in cow urine.     

Investigation on possible transformations of cortisol,cortisone and cortisol glucuronide in bovine faecal matter using liquid chromatography–mass spectrometry

Given the close resemblance of the ring A structure of prednisolone and prednisone on the one hand, and of androstadienedione on the other, the transformation of cortisol and cortisone into prednisolone and prednisone in cattle faeces was evaluated. A simple method that does not involve extraction but only the 1:100 dilution of cattle faeces, spiking with 400 ng/mL cortisol, cortisone or cortisol glucuronide and incubation of the suspension, was used. The analyses were performed by HPLC–MS³ to detect the supposed Δ¹ dehydrogenation of the glucocorticoids. The decision limits (CCα) and detection capabilities (CCβ) were 2.0 and 3.0 ng/mL for cortisol, cortisone and prednisolone, 3.0 and 4.0 ng/mL for cortisol glucuronide and 7.0 and 10.0 ng/mL for prednisone, respectively. Intra-day and inter-day coefficients of variation (CV%), were 5.6–6.2 and 5.2–6.6 for cortisol glucuronide, cortisol, cortisone and prednisolone, and 16.0 and 16.2 for prednisone, respectively. The recoveries were in the range 110–143% for all analytes. Regression coefficients (R2) were in the range 0.996–0.999 for all analytes. The results show the hydrolysis of the conjugated form and the dehydrogenation in ring A in diluted faeces. It is therefore predicted that urine contaminated with faeces may be positive for prednisone and prednisolone in the same way as they are positive for boldenone, i.e. as a result of microbiological dehydrogenase activity on cortisol and cortisone.     

Development of a sensitive and selective method for the quantitative analysis of cortisol,cortisone, prednisolone and prednisone in human plasma

A highly selective, sensitive and robust LC–MS/MS method was developed for the simultaneous quantification of cortisol, cortisone, prednisolone and prednisone in human plasma. Prednisolone, cortisol and cortisone have similar fragmentation pattern. These three compounds were chromatographically separated, thus eliminating the inherent interference that fragments derived from the M + 2 and M isotopes of prednisolone contribute in the MRM channels of cortisol and cortisone, respectively. Additionally, by using a small particle (1.8 μm) analytical column, interferences present in the plasma samples from post-transplant recipients were successfully resolved from cortisol after a simple extraction consisting of protein precipitation, evaporation and reconstitution. The chromatographic separation was achieved on a Zorbax-SB Phenyl column under isocratic conditions during a run time of 8 min. Intra-run and inter-run precision and accuracy within ±15% were achieved during a 3-run validation for quality control samples at five concentration levels in charcoal-stripped plasma as well as in normal plasma, over a 500-fold dynamic concentration range. The lower limit of quantitation was 0.500 ng/mL for cortisone and prednisone, 1.00 ng/mL for cortisol and 2.00 ng/mL for prednisolone. The performance of the small particle column was maintained during more than 1200 injections in terms of peak retention time, symmetry and backpressure.     

Simultaneous determination of prednisolone, prednisone, cortisol, and cortisone in plasma by GC-MS: estimating unbound prednisolone concentration in patients with nephrotic syndrome during oral prednisolone therapy

Individual variability of the pharmacokinetics of prednisolone based on the unbound concentration in plasma is of significant clinical consideration. The unbound concentrations of prednisolone were measured in 10 patients with nephrotic syndrome, two patients with systemic lupus erythematosus, and one patient with dermatomyositis by examining protein bindings of prednisolone on one or more occasions during prednisolone treatment. In this study, plasma concentrations of prednisolone, prednisone, cortisol, and cortisone were simultaneously analyzed by GC-MS by using stable isotope-labeled internal standards. Equilibrium dialysis was employed to accurately estimate the unbound fractions of prednisolone in plasma. The unbound fraction of prednisolone changed depending on plasma total prednisolone concentration and plasma albumin concentration. The unbound fraction of prednisolone (Y) is calculated: Y=(-0.0101x' + 0.0736) x + 10.23, where x' is the plasma albumin concentration and x is the total prednisolone concentration. The estimated concentrations of unbound prednisolone by using the above equation were in good agreement with the measured concentrations of unbound prednisolone. Since the protein binding of prednisolone did not change in the presence of prednisone (114.0 ng/ml), it appeared that prednisone produced from the therapeutic dose of prednisolone did not affect the unbound fraction of prednisolone.     

Simple liquid chromatography method for the rapid simultaneous determination of prednisolone and cortisol in plasma and urine using hydrophilic lipophilic balanced solid phase extraction cartridges

This article describes the development and validation of a simple solid phase extraction (SPE) and HPLC method for the extraction and the specific determination of prednisolone and hydrocortisone (cortisol) in both plasma and urine using one washing step with Oasis hydrophilic lipophilic balanced (HLB) cartridges (1 ml/30 mg, 30 microm). Recoveries of prednisolone and cortisol from plasma and urine exceeded 82%. The limit of quantification (LOQ) in plasma and urine was 9.9 and 6.7 ng/ml for cortisol, respectively, and 11.6 and 8.0 ng/ml for prednisolone, respectively. The intraday and interday precision (measured by CV%) for both prednisolone and cortisol in both plasma and urine was always less than 7%. The accuracy (measured by relative error %) for both prednisolone and cortisol in both plasma and urine was always less than 8%. The advantages of the developed method are the use of a one step washing SPE utilising HLB cartridges which do not suffer the drying out problems of conventional SPE cartridges and the time saving when compared with solvent extraction (SE), in addition to the simultaneous determination of prednisolone and cortisol in both plasma and urine.     

Simultaneous determination of prednisolone, prednisolone acetate and hydrocortisone in human serum by high-performance liquid chromatography

A method for the simultaneous determination of prednisolone, prednisolone acetate and hydrocortisone has been established to monitor the serum levels of these three compounds in healthy volunteers following intramuscular administration of prednisolone acetate. Serum samples of 0.75 ml were extracted with ethyl acetate after addition of the internal standard, dexamethasone. The compounds were separated using a LiChrosorb Si 60 column and detected by UV absorbance. Specificity, linearity, as well as the repeatability, intermediate-precision and accuracy of the method were established. The lower limit of quantification was 2.0 ng/ml for prednsolone (C.V. = 14.7%, N=6) and 5.0 ng/ml for prednisolone acetate (C.V. = 13.9%, N= 6 and hydrocortisone (C.V. = 11.7%, N=6). Data on the recovery of the compounds and the internal standard are provided. The results of quality control samples determined during routine analysis (n = 114) are presented. Serum levels of the compounds after intramuscular adminstration of 25 mg of prednisolone acetate are discussed.     

Liquid chromatographic analysis of prednisolone, prednisone and their 20-reduced metabolites in perfusion media

A reversed-phase liquid chromatographic assay was developed to quantitate prednisolone, prednisone and the 20α-dihydro and 20β-dihydro reduced metabolites of both parent compounds in tissue culture media from in vitro perfusions of the human placental lobule. Steroids were extracted from perfusate, using reversed-phase cartridges, with average recoveries of 95.2% or greater. The internal standard for the analyses was 6α-methylprednisolone. In this assay cortisol coelutes with prednisolone, however, no other significant interferences were found. Assay of each steroid was linear in the range 0–1 μg/ml. Intra-assay coefficients of variation were measured at 10 and 750 ng/ml with ranges of 3.4% (20α-dihydroprednisone) to 8.8% (20β-dihydroprednisolone) and 4.1% (20β-dihydroprednisone) to 8.8% (prednisone). The corresponding inter-assay coefficients of variation were 3.3% (20α-dihydroprednisone) to 9.1% (20β-dihydroprednisolone) and 1.9% (prednisolone) to 3.5% (prednisone). The analyses utilized two C₁₈ columns which were linked together and maintained at 40°C.     

Simultaneous determination of dexamethasone and 6 beta-hydroxydexamethasone in urine using solid-phase extraction and liquid chromatography: applications to in vivo measurement of cytochrome P450 3A4 activity

It has been demonstrated that the formation of the hydrophilic metabolites of dexamethasone, 6 alpha- and 6 beta-hydroxydexamethasone, correlated with cytochrome P450 (CYP) 3A4 enzyme levels. So, the 6 beta-hydroxydexamethasone/dexamethasone urinary ratio could be a specific marker for human CYP3A4 activity. We have developed a sensitive and specific high-performance liquid chromatographic method for the simultaneous quantification of urinary free dexamethasone and 6 beta-hydroxydexamethasone using 6 alpha-methylprednisolone as internal standard. This method involved a solid phase extraction of the three compounds from urine using Oasis HLB Waters cartridges with an elution solvent of ethyl acetate (2 ml) followed by diethyl ether (1 ml). Separation of the three analytes was achieved within 24 min using a reversed-phase Nova-Pak C(18) analytical column (4 microm, 300 mm x 3.9 mm i.d.). An ultraviolet detector operated at 245 nm was used with a linear response observed from 10 to 100 ng/ml for dexamethasone and from 25 to 1000 ng/ml for 6 beta-hydroxydexamethasone. Obtained from the method validation, inter-assay precision was below 15% and accuracy ranged from 95.7 to 110%. The extraction efficiency of the assay was approximately of 99% and was constant across the calibration range. The lower limit of quantitation was 10 ng/ml for dexamethasone and 25 ng/ml for 6 beta-hydroxydexamethasone; at these levels, precision was below 16% and accuracy was 99-109%. This method was applied to in vivo measure of the CYP3A4 activity.     

Corticosteroid analysis in biological fluids by high-performance liquid chromatography

A sensitive, specific, and reproducible high-performance liquid chromatographic assay for the simultaneous determination of prednisone, prednisolone and cortisol in biological fluids was developed with dexamethasone as the internal standard. Samples are extracted with methylene chloride, washed with sodium hydroxide and then water, and chromatographed on a microparticulate silica gel column with UV detection at 254 nm. Sensitivity was greater than 15 ng for all four steroids. Specificity was supported by use of dual wavelength UV detection and/or radioimmunoassay. The assay has been applied in pharmacokinetic studies and a typical plasma concentration—time profile for the three steroids is presented for one subject who received 50 mg of prednisone.     

Dexamethasone-suppressible hypercorticosteronism in two 46,XX subjects with ambiguous genitalia and ovarian cysts. Partial defect of 17 alpha-hydroxylase or 17-20-desmolase

The paradoxical association of female pseudohermaphroditism and androgen deficiency was observed in two 46,XX subjects with high corticosterone plasma levels. Subject 1 has been declared a boy due to clitoris enlargement; she had no vagina and uterus. Subject 2 had ambiguous external genitalia. In both, at age 27 and 17 years, fusion of outer labia, impuberism, ovarian cysts, and histologically normal ovarian tissue were observed. Blood pressure was normal. Basal cortisol levels were normal but unresponsive to ACTH. Progesterone levels were 40 and 62 ng/ml and rose after ACTH (50 and 79 ng/ml). 17-hydroxyprogesterone levels were 25 and 21 ng/ml and did not rise after ACTH. Corticosterone levels were 70 and 92 ng/ml and rose after ACTH (110 and 180 ng/ml). All three steroids were suppressed by dexamethasone. Androgen and estrogen levels were at or below the lower limit for normal women. The sex steroid levels obtained by radioimmunoassay in plasma and a follicular cyst fluid were confirmed by isotope dilution-mass spectrometry. We suggest that the sexual ambiguousness resulted from an excessive production of gestagenic steroids during fetal life, and that the enzyme defect is either a partial 17 alpha-hydroxylase defect combined with a peripheral production of 17-hydroxyprogesterone, or else a partial 17-20-desmolase defect with a secondary 21-hydroxylase defect limited to the cortisol pathway.     

Simultaneous determination of cortisol, dexamethasone, methylprednisolone, prednisone, prednisolone, mycophenolic acid and mycophenolic acid glucuronide in human plasma utilizing liquid chromatography-tandem mass spectrometry

Chronic combination immunosuppressive regimens are commonly prescribed to renal transplant recipients. To develop an assay method for pharmacokinetic studies and therapeutic drug monitoring of multiple immunosuppressives, a liquid chromatography-tandem mass spectrometry (LC/MS/MS) approach for the simultaneous analysis of several glucocorticoids, mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG) was investigated. The resultant method utilized a gradient reverse phase separation over a Symmetry C18 column using an ammonium acetate-methanol mobile phase at pH 3.5. The analytes were detected by coupling the chromatography system via electrospray to a triple quadrupole mass spectrometer. Multiple-reaction monitoring in the negative mode ion (MH-/product) was employed selecting MPA at 319.1/190.9, MPAG at 495.1/191.0, dexamethasone at 391.0/361.0, hydrocortisone at 361.1/331.1, methylprednisolone at 373.1/343.1, prednisone at 357.1/327.2, and prednisolone at 359.1/329.1. The calibration curve concentrations ranged from 3.60 ng/mL to 50 microg/mL with the lowest limit of quantitation for corticosteroids being 3.60-7.20 ng/mL and 0.656-6.75 microg/mL for MPA and MPAG, respectively. The relative standard deviation for quality control intraday variation and interday variation was between 0.76% and 9.57% for all analytes. This assay offers a versatile, unique method for multi-analyte immunosuppressive determinations during combination immunosuppression.     

Simultaneous determination of glucocorticoids in plasma or urine by high-performance liquid chromatography with precolumn fluorimetric derivatization by 9-anthroyl nitrile

A new method for simultaneous determination of glucocorticoids (GCs) in plasma or urine by high-performance liquid chromatography (HPLC) with fluorimetric detection has been developed. Following extraction with ethyl acetate using a reversed-phase disposable cartridge, the six GCs [cortisol (F), cortisone (E), prednisolone (PL), prednisone (PN), 6β-hydroxycortisol (6β-OHF) and 6β-hydroxyprednisolone (6β-OHP)] and an internal standard (6β-hydroxycotortisone) were derivatized by treatment with 9-anthroyl nitrile (9-AN) in a mixture of basic catalysts (triethylamine and quinuclidine) to give the fluorescent esters through the 21-hydroxyl group. The GC derivatives so obtained were then cleaned by a straight-phase disposable cartridge and chromatographed on a straight-phase column with an isocratic HPLC technique. The fluorescence derivatives of the GCs, including the internal standard, were separated as clear single peaks and no interfering peaks were observed on the chromatograms. The lower limits of detection for F, E, PL and PN in plasma or urine were 0.1 ng/ml and those for 6β-OHF and 6β-OHP in plasma or urine were 0.5 ng/ml, at a signal-to-noise ratio of 3. The analytical recovery of known amounts of the GCs added to plasma or urine were almost 100%. This method can be applied to the determination of plasma or urinary F in renal transplant patients who received PL and can be applied for other metabolic investigations in relation to the change in blood pressure via 11β-hydroxysteroid dehydrogenase or in hepatic metabolizing via CYP3A4.     

Analysis of prednisone,prednisolone and their 20β-hydroxylated metabolites by high-performance liquid chromatography

A high-performance liquid chromatographic technique primarily developed for use on samples from kidney perfusion studies is presented for simultaneous determination of prednisone, prednisolone and their 20β-hydroxylated metabolites. The technique employs 6β-hydroxycortisol as the internal standard. Samples are extracted with ethyl acetate, washed with sodium hydroxide and water and injected onto a silica gel column with UV detection at 254 nm. Inter- and intraday variability of the assay was determined at two concentrations of each steroid and was less than 10%. Assay steroid recovery ranged from 54.1% for prednisone to 63.2% for 20β-hydroxyprednisone. Sensitivity is 4–10 ng/ml for the steroids measured. The chromatographic conditions may be modified to permit quantitation of these steroids from plasma samples. This method may alternatively be used for quantitation of 6β-hydroxycortisol, an endogenous indicator of enzyme induction. A perfusate concentration-time profile is presented from a kidney perfusion study using prednisolone.     

Serum cortisol concentrations during low dose dexamethasone suppression test to screen for Cushing's syndrome.

Forty four subjects (23 obese controls, 11 patients with possible Cushing''s syndrome, and 10 patients with definite Cushing''s syndrome) underwent low dose (0 X 5 mg every six hours for two days) dexamethasone suppression tests during which serum cortisol concentration at 0800 and excretion of urinary free cortisol over 24 hours were measured. Serum cortisol concentration fell to below 60 nmol/1 (2 X 2 micrograms/100 ml) in 31 subjects and remained above 250 nmol/1 (9 X 1 micrograms/100 ml) in the 13 others. Excretion of urinary free cortisol showed a similar response, falling to below 110 nmol (40 micrograms)/24 h in 31 and remaining above 180 nmol (65 micrograms)/24 h in the 13 others. There was complete concordance between the two variables in terms of the pattern of response. Serum cortisol concentration fell to below 60 nmol/1 (2 X 2 micrograms/100 ml) in at least 97% (31 of a possible 32) of subjects without Cushing''s syndrome. On the other hand, a serum cortisol concentration of above 250 nmol/1 (9 X 1 micrograms/100 ml) after low dose dexamethasone gave a false positive diagnosis of Cushing''s syndrome in at most only one of 13 patients (7 X 7%). Measurement of serum cortisol concentration during the low dose dexamethasone test is simpler than, and as accurate and reliable as, measurements of urinary steroids.     

Pharmacokinetics of prednisone and prednisolone in a case of hypothyroidism: effect of replacement therapy

We found this particular case during the course of a clinical trial designed to assess the pharmacokinetics of oral prednisone in normal and diseased children. The plasma concentrations of prednisone, its main metabolite prednisolone, and endogenous cortisol were measured by HPLC at selected times during 8-h periods starting at 7:30 a.m. One 9.9-year-old administered prednisone 0.5mg/kg p.o. was found to be hypothyroid (TSH: 351microIU/mL; fT4: <2pg/mL; fT3: <1pg/mL); four age-matched normal boys (aged 6.6+/-4.9 years) served as a control group. In comparison with the controls, the hypothyroid boy showed a marked increase in the total AUC of prednisone (3360microg h/L versus 215+/-83microg h/L) and prednisolone (4040microg h/L versus 724+/-77microg h/L), and an altered pattern of endogenous cortisol, which is known to be impaired in hypothyroid subjects. After 6 months of thyroxine replacement therapy (75microg/day), the AUCs of prednisone and prednisolone returned to normal values (prednisone: 248microg h/L; prednisolone: 528microg h/L), as did the pattern of circadian cortisol secretion. In conclusion, our data indicate that the pharmacokinetics of prednisone and prednisolone can be profoundly altered by hypothyroidism, and subsequently restored by thyroxine replacement therapy.     

A simple method for separating unbound and bound cortisol in a radioimmunoassay

A cortisol radioimmunoassay in which unbound cortisol is partitioned into the organic phase of a toluene: water scintillation fluid mix at 0 to 5 degrees C is described. Antibody-bound cortisol remained in the aqueous phase. Since liquid scintillation spectrometers detect photons generated from the [3H]cortisol only in the organic phase, the system effectively separates antibody bound from unbound [3H]cortisol. Regression coefficients including linear, quadratic, and cubic components of standard curves were between 0.980 and 0.999. Cross-reactivity was 3% or less with 11 other steroids and cholesterol except for cortisone (16%) and prednisone (12%). Intra- and interassay coefficients of variation were 8 and 13%, respectively. The lower limit of sensitivity of the assay was 1.4 ng/ml. Recoveries of added mass averaged 97.5%. The correlation between concentrations of glucocorticoids assayed by competitive binding to dog plasma and the current procedure was 0.90. The assay procedure described simplifies separation of unbound from antibody-bound cortisol.     

Serum concentrations of deoxycorticosterone in women during the luteal phase of the ovarian cycle are not suppressed by dexamethasone treatment

We determined the serum levels of deoxycorticosterone (DOC) in plasma of six healthy, apparently ovulatory women during the mid-follicular and mid-luteal phases of their ovarian cycles; and we evaluated the effect of dexamethasone (1 mg by mouth) on the concentrations of DOC and cortisol in serum at times when plasma progesterone levels were high or low. The serum levels of DOC, unlike those of cortisol, did not vary significantly in single blood samples obtained in the morning (8-10 a.m.) and afternoon (3-5 p.m.); and serum DOC levels in women were significantly higher (P less than 0.05) during the mid-luteal phase than during the mid-follicular phase of the cycle. There were unmistakable diurnal variations in serum levels of cortisol, and cortisol concentrations were reduced to less than 20% of pretreatment levels after the ingestion of 1 mg dexamethasone during the mid-follicular or mid-luteal phase. The serum concentrations of DOC were reduced only to approx 70% of pretreatment levels after dexamethasone ingestion during the follicular phase. The serum levels of DOC did not decline significantly after administration of dexamethasone during the mid-luteal phase, when progesterone levels in serum are high (14-16 ng/ml). Blood samples also were obtained at hourly intervals during the 24 h before and after dexamethasone administration in one woman during the follicular phase and in another woman the during the early luteal phase (progesterone levels = 1-3 ng/ml) of the ovarian cycle. DOC levels (pre-dexamethasone) fluctuated in synchrony with those of cortisol in the woman studied during the follicular phase but not in the woman studied during the early luteal phase of the cycle. In the post-dexamethasone period, plasma cortisol levels were suppressed for at least 24 h in both women whereas DOC levels were decreased only partially. We conclude that plasma DOC is derived from both adrenal secretion and from extraadrenal 21-hydroxylation of progesterone--the latter source of DOC is not affected by dexamethasone suppression of ACTH secretion.     

Improved liquid chromatographic determination of serum cortisol with double internal standardization compared to radioimmunoassay and fluorometry, and evaluated by isotope dilution/mass spectrometry

A sensitive and specific high-performance liquid chromatographic (HPLC) method for the determination of cortisol in only 200 microliters of serum is described. Cortisol and two internal standards, 19-nortestosterone (IS1) and 6 alpha-methylprednisolone (IS2) are extracted with dichloromethane and analyzed on a C18 reversed-phase column eluted with a mobile phase of methanol:water at a flow rate of 0.75 ml/min. Ultraviolet absorption at 254 nm is used for detection and quantitation is performed by peak height ratio measurement. Using 200 microliters of serum, the lower limit of detection for cortisol is 10 ng/ml, the analytical recovery is 104 +/- 3.6% (n = 8), and the day-to-day precision was 1.69% at a level of 90 ng/ml (n = 16). Cortisol values obtained by this method were generally lower than those obtained by radioimmunoassay or by fluorometry. A serum pool was analyzed both by HPLC and by isotope dilution/mass spectrometry (ID/MS). A mean value of 90.1 ng/ml was obtained by HPLC (n = 16, CV = 1.7%), whereas ID/MS yielded a mean of 90.8 ng/ml (n = 28, CV = 0.4%). These results clearly demonstrate the high specificity and the accuracy of the HPLC procedure. The use of two internal standards not only compensates for losses during the sample manipulation but also prevents erroneous results in case of medication by either of these two products.     

On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. III. Determination of prednisolone in serum

Solid-phase extraction (SPE) was directly coupled to mass spectrometry (MS) to assess the feasibility of the system for the rapid determination of prednisolone in serum. A C(18) stationary phase allowed washing of the cartridge with 25% methanol. Elution was performed by switching the methanol percentage from 25% in the washing step to 50% during elution. The high flow-rates during the extraction (5.0 ml/min) combined with ion-trap MS detection resulted in a total analysis time of 4 min. Some tailing of the prednisolone peak was observed. However, the tailing was found acceptable, since by this elution procedure most matrix compounds were prevented from eluting from the cartridge. Some matrix interference was still observed with a triple-quadrupole MS, even in the multiple reaction monitoring mode. This resulted in a detection limit (LOD) of about 10 ng/ml. The matrix interference and the LOD were similar for atmospheric pressure chemical ionisation and atmospheric pressure photo ionisation. Applying an ion-trap MS in the MS-MS mode resulted in cleaner chromatograms. Due to extensive fragmentation of prednisolone, the LOD was not lower than about 5 ng/ml prednisolone in serum, and a limit of quantitation of about 10 ng/ml (relative standard deviation <15%) was observed.