C269 Walker Figure 1 formatted Submitted crop

UPLC-MS/MS measurement of prednisolone in adrenal insufficiency

Prednisolone is an attractive once-daily option to treat adrenal insufficiency. Its prior association to osteoporosis and diabetes is possibly due to widespread over-replacement. With the availability of an ultra-performance liquid-chromatography tandem mass spectrometry (UPLC-MS/MS) method to detect serum concentrations and guide treatment, we can assess the true effects of long-term low-dose prednisolone therapy.

by Dr Sirazum Choudhury and Dr Emma Williams

Prednisolone is a pioneering synthetic corticosteroid synthesized by Arthur Nobile in 1950 as an anti-arthritic treatment [1, 2]. Sharing a similar structure to cortisol, prednisolone benefits from a longer half-life and increased potency compared to endogenous steroids, owing to a double bond found between C1 and C2 on the first carbocyclic ring (Fig. 1). Prednisolone has proven to be an indispensable anti-inflammatory drug and has long been used in the treatment of many conditions including asthma, inflammatory bowel disease and rheumatoid arthritis.

Use of prednisolone for adrenal insufficiency
More recently prednisolone is gaining traction as an option for glucocorticoid replacement therapy in adrenal insufficiency. There are an estimated 8400 individuals living with the condition in the UK, with an annual incidence of 4.4–6 cases per million in Europe [3]. The challenges of adrenal insufficiency are well characterized. In the era prior to the availability of effective treatment, the associated mortality was 85% in 2 years, and up to 100% in 5 years [4]. Over the last half-century, our increasing understanding of steroids has meant that patients are living longer, with a life expectancy approaching that of the normal population. However, a mortality gap does remain, which may in part be due to incorrect replacement of glucocorticoids, concurrently increasing the risk of diabetes, osteoporosis and cancer.

Oral hydrocortisone is the most commonly prescribed treatment for adrenal insufficiency, but is perhaps not the most ideal [5]. Due to the relatively short half-life, hydrocortisone must be administered three times daily, which can hinder compliance. For this reason it is our experience that some patients tend to omit the last dose of the day. Moreover, the price of hydrocortisone has been rising in the UK, costing £76 for a 1-month supply of 10 mg. This contrasts to a 1-month supply of 5 mg prednisolone tablets, which costs £0.88. With prednisolone offering a once-daily solution to adrenal insufficiency, it now features in the Endocrine Society clinical practice guidelines from earlier this year as an alternative to thrice-daily hydrocortisone therapy [6].

Prednisolone dose and adverse effects
The biggest obstacle to the widespread acceptance of prednisolone as a viable therapy has been its association with adverse metabolic effects such as osteoporosis. This is as a result of multiple studies purporting to show that ‘low dose’ prednisolone has a negative impact of the markers of bone turnover and bone absorptiometry [7]. Based on an assumed bioequivalence ratio of 4 : 1, 7.5 mg of prednisolone was judged to be equivalent to 30 mg of hydrocortisone and was considered ‘low dose’. The basis of this ratio is difficult to ascertain but was probably calculated from data on anti-inflammatory doses of prednisolone, which are significantly higher than the doses likely to be needed in steroid replacement therapy. More recently, a study comparing prednisolone to hydrocortisone in 44 children with congenital adrenal hyperplasia found that a lower dose of prednisolone than expected was required to control the condition [8]. Using objective biological markers, such as growth velocity, and hormonal markers such as androstenedione and 17-hyroxyprogesterone, the group discovered that prednisolone is 1.5 to 2 times more potent than previously thought, suggesting that a more appropriate prednisolone replacement dose is in fact 3 mg to 5 mg, and not as high as 7.5 mg.

To facilitate this shift towards the use of even lower doses of prednisolone, it is important to provide reassurance to both clinicians and patients that the lowest necessary dose of prednisolone is used to maintain an appropriate trough level towards the end of the day. This would be in keeping with the diurnal rhythm of cortisol. Our ability to more accurately and efficiently report serum prednisolone concentrations using an ultra-performance liquid-chromatography (UPLC) tandem mass spectrometry (MS/MS) technique provides this confidence.

Measurement of plasma prednisolone concentrations
Historically, the first assays to measure plasma prednisolone concentrations were competitive protein binding assays and radio-immunoassays [9]. The protein binding assays were designed to use cortisol binding globulin and were therefore non-specific to prednisolone. The early radio-immunoassays were prone to interference from other endogenous steroids and intermediaries, making them unreliable especially if patients continued to produce subclinical levels of cortisol. Specificity could be improved with the addition of a thin layer chromatography preparatory step; however, the lower limit of detection remained as high as 20 µg/L.

In the 1970s, high-performance liquid-chromatography (HPLC) methods gained popularity [10]. Offering greater specificity for prednisolone, the method involved a time-consuming liquid–liquid-extraction sample-preparation step. The extracted organic phase would be dried before being reconstituted with mobile phase and passed through a normal phase hydrophilic interaction chromatography HPLC column. Prednisolone concentrations were detected with ultraviolet absorbance spectrophotometry. Although this method could identify different corticosteroids, it proved to be cumbersome with retention times of up to 8 minutes for prednisolone, and 20 minutes for other steroids. With 76% recovery and a lower limit of detection of 25 µg/L, this technique is not suitable to assess trough levels of prednisolone, with a high likelihood of reporting undetectable results at the lower end, potentially facilitating over-replacement in patients.

Using a UPLC-MS/MS method, we are able to overcome the obstacles that have plagued prednisolone assays in the past (reference awaiting PubMed identifier). Serum samples are prepared using a protein precipitation method, involving zinc sulphate and the addition of deuterated (D6) prednisolone as internal standard. Following preparation, the extract is combined with both methanol and water based mobile phases before being passed through a C-18 chromatography column, which employs a reversed phase partition process. Prednisolone is eluted at approximately 1.0 minutes, before being detected by multiple reaction monitoring using electrospray ionization in positive ion mode. An example of the observed chromatograms can be found in Figure 2.

This method of measuring plasma prednisolone concentrations is linear to prednisolone concentrations of 1000 µg/L (Fig. 3), with an inter- and intra-assay co-efficient of variance at 50 µg/L of 4.1% and 2.5% respectively. The technique has proven more sensitive than HPLC with the lower limit of quantification at 10 µg/L without the HPLC recovery issues, and is equally as specific to prednisolone. By using a protein precipitation method, the preparation step is now significantly shorter. Additionally, with reduced prednisolone retention times, a prepared sample can now be analysed in 3.5 minutes before the next sample is immediately run. As a result, the UPLC-MS/MS technique is better suited to the modern clinical biochemistry laboratory being able to reliably cope with larger numbers of patient samples in shorter times than previously thought possible.

Measuring serum prednisolone concentrations has proven extremely valuable in monitoring glucocorticoid replacement therapy. There is observable variability in prednisolone metabolism between individuals, with terminal half-lives routinely varying between 1.75 and 3.75 hours. We currently measure a trough level at 8 hours post-prednisolone administration aiming for a concentration of 10–20 µg/L to ensure adequate replacement throughout the day and preserve an overnight corticosteroid nadir. The results are used clinically to inform the decision either to increase or decrease prednisolone doses as appropriate but also serve as objective proof to patients who are anxious about a reduction. The assay is also clinically useful in confirming patient compliance with their prescribed medication.

Future perspectives
Beyond the clinical utility in quantifying serum prednisolone levels, there is significant research potential for this assay. Addisonian crises are currently responsible for up to 15% of deaths in patients with adrenal insufficiency [11]. Our understanding of the disease process is limited by the urgency to provide treatment with either intravenous or intramuscular hydrocortisone, before a blood sample is taken. As this is detected by cortisol assays, it is difficult to interpret whether the pre-crisis hydrocortisone concentration was inadequate (suggesting non-compliance or reduced absorption) or appropriate (suggesting that the level was insufficient to match requirement). In patients treated with prednisolone who present with Addisonian crises, the assay will allow us to assess the pre-treatment serum prednisolone concentrations, even if the blood sample is taken after treatment with hydrocortisone.

More importantly in the immediate setting, it is anticipated that the previously accepted long-term effects of ‘low dose’ prednisolone can be explored. The availability of a reliable and specific assay will result in a greater number of patients on prednisolone who are appropriately treated and not over-replaced. In time, as more data becomes available, we will gain a clearer picture of the true effects of prednisolone.

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The authors
Sirazum Choudhury BSc, MBBS, MRCP
and Emma Williams* BSc, PhD, FRCPath
Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK

*Corresponding author
E-mail: emma.walker@imperial.nhs.uk