LC-MS candidate reference methods for the harmonisation of parathyroid hormone (PTH) measurement: a review of recent developments and future considerations

Assay variability – causes, concerns and the need for harmonisation

The analysis of ‘true’ PTH1-84 is not straightforward – there exists a surprisingly heterogeneous range of PTH-related peptides [6]. As will be discussed, both the exact composition and possible biological functions of PTH fragments remain to be fully elucidated as does the variable influence of these fragments on currently available analytical methods for PTH testing [2, 7]. PTH1-84 can be proteolytically cleaved within the parathyroid gland prior to secretion, or cleavage can occur through the actions of endoproteases in hepatic Kuppfer cells [8, 9]. Certain synthetic PTH fragments have been demonstrated to antagonise the biological activity of PTH1-84 both in vitro and in vivo [10, 11]. The discovery of circulating PTH fragments in the late 1960s and 1970s was the main driver for the development of second- and third-generation immunoassay methods for PTH analysis.

It is perhaps ironic that it is in patients with renal failure, in whom the accurate analysis of PTH1-84 is clinically important for the prevention of mineral bone disorders and for guiding expensive drug treatments, that the accumulation of PTH fragments is the most pronounced. A review of circulating PTH isoforms based on immunoassay by D’Amour [11] concluded that PTH1-84 accounts for just 20% of the ‘total’ circulating PTH in normocalcaemic individuals, and can be as little as 5% in patients with end-stage renal failure, depending upon which assay is used for measurement.

Numerous publications have highlighted discrepancies between ‘PTH1-84’ results from the same sample analysed using different analytical platforms [2, 7, 12]. Indeed, it has been proposed that the difference between two different immunoassay results could provide a useful index for the diagnosis and prognosis of parathyroid carcinomas [13–15].

There is an obvious clinical need for the harmonisation of PTH testing. The variability which exists in PTH testing should be considered a critical governance issue in patients with chronic kidney disease [16]. Given this variability in results produced using different methodologies, some clinicians question the relevance of measuring PTH at all in renal patients [2, 17]. Often, assay preference is governed more by reliance upon a method with which they have the most familiarity and clinical experience than by absolute analytical accuracy [8]. It is hoped by many that the establishment of an International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) working group for PTH [18] will aid the development of reference methodologies, as well as harmonise the testing of PTH towards a traceable reference material (e.g., the World Health Organization Recombinant PTH1-84 International Standard 95/646, available through the National Institute for Biological Standards and Control, NIBSC – www.nibsc.org). Until such time as a reference method is developed and validated, which can be used to establish target values based on reference materials, the IFCC group are working towards the development of assay-specific target values (more information on the IFCC working group is available through http://www.ifcc.org/ifcc-scientific-division/sd-working-groups/parathyroid-hormone-wg-pth/).

The following sections aim to review the current literature on the development of PTH assay methods, with particular emphasis on the influence of circulating PTH fragments on the accuracy of analytical methods designed to measure intact PTH1-84. The use of mass spectrometry (MS)-based approaches for both the identification of PTH fragments and for the development of quantitative PTH assays will be reviewed, and important considerations for the development of analytical methods that are harmonised towards a standardised reference method will also be discussed.

Second-generation immunoassays

It was the discovery of N-terminally truncated PTH fragments (e.g., PTH34-84) at high concentrations in patients with terminal renal failure, plus the knowledge that the biological activity of PTH1-84 resided within the N-terminal region, that led to the development and introduction of second-generation (or ‘intact’), two-site immunoassays [21, 22]. These intact PTH assays typically have a solid-phase capture antibody directed towards the C-terminal region of PTH (amino acids 39-84), and a detection antibody directed towards the N-terminus (Figure 3), usually towards amino acids 12-24, though a few second-generation assays were developed with a detection antibody epitope directed towards the 26-32 region [23–25]. These assays were thought to be far more specific, since they avoided cross-reactivity with C-terminal PTH fragments. They were certainly an improvement upon the early single-antibody, mid-region, C-terminal RIAs as judged by correlation with clinical findings [26, 27]. In this form, these assays were widely adopted by many clinical laboratories, reference ranges for different patient groups were developed, and indeed, they are still used by many to this day for routine PTH testing.

Figure 3

Antibody epitopes of first-generation RIAs, second-generation (‘intact’) and third-generation (‘whole’) PTH immunoassays.

Third-generation immunoassays

Based on findings from a parathyroid function study in 1993 [28], Brossaud et al. [29] developed a chromatographic method to separate C-terminal PTH fragments from PTH1-84 prior to immunoassay analysis (second-generation). They reported that, in addition to the known C-terminal PTH fragments missing the entire N-terminus (e.g., PTH34-84), there existed a second group of larger C-terminal PTH fragments with a partially preserved N-terminus, which accumulated in renal failure. They later established that the fragmentation occurred somewhere within the first 19 amino acids of the PTH N-terminus based on comparison of second-generation immunoassays with different detection antibody epitopes [24]. These fragments were collectively described as ‘non-1-84’ PTH fragments, and were demonstrated to cross-react in second-generation immunoassays using recombinant PTH7-84, a commercially available peptide, as a surrogate for this group of peptides. In renal failure, the cross-reactivity of these non-1-84 fragments was shown to account for up to 60% of total immunoreactive PTH, depending upon which immunoassay was used [11, 23–25, 29, 30]. These observations supported clinical findings that second-generation immunoassay results did not always correlate with other clinical and biochemical results [31, 32].

This led to the development of third-generation (‘whole PTH’) assays [33, 34], in which the detection antibody epitope was targeted further towards the N-terminus of PTH1-84, to amino acids 1-4, thus avoiding cross-reactivity with the newly observed large C-terminal PTH fragments (Figure 3). Despite the improved performance of these assays in some patient groups [35], some anomalous results were still reported [36–38].

‘Amino-PTH’

D’Amour and co-workers explored the exact structure of non-1-84 PTH fragments, using the array of second- and third-generation immunoassays that were then available, plus Edman degradation radio-sequencing with PTH with ³⁵S-labelled methionine residues. Samples from healthy controls were compared with individual clinical samples and sample pools from patients with confirmed parathyroid pathology and/or renal failure. Ex vivo tissue samples from patients with confirmed primary and secondary hyperparathyroidism were also used to generate cell lines in vitro. Findings included first, that PTH7-84 was likely to be the most abundant form of the non-1-84 PTH cross-reacting in second-generation immunoassays; second, fragments corresponding to PTH4-84, PTH8-84, PTH10-84 and PTH15-84 were also present; and third, that another non-1-84 PTH fragment, over-expressed in severe hyperparathyroidism and parathyroid cancers, cross-reacted with third-generation but not second-generation immunoassays with a 12-20 epitope [11, 24, 25, 30]. It was suggested that this PTH variant (termed ‘amino-PTH’ in some references), which appears to have an intact N-terminus, may represent a modified variant form of PTH, with a modification in the 15-20 region. This would explain its lack of cross-reactivity with second-generation immunoassays [23, 39].

Mass spectrometry

In the last 10–15 years, liquid chromatography coupled with mass spectrometric detection (LC-MS or tandem MS, LC-MS/MS) has become a widely applied technique in clinical chemistry laboratories. It has largely been used for the quantitative analysis of small-molecules (i.e., <1000 Da), such as steroids and related hormones (e.g., testosterone and vitamin D metabolites) and drugs and their metabolites. Modern LC-MS instruments are highly sensitive, and coupled with appropriate sample preparation techniques, can often achieve limits of detection comparable with immunoassays (e.g., pmol/L for some analytes). However, it is the combination of chromatographic separation with a detection system based on mass-to-charge (m/z) ratio, which provides a specificity of LC-MS that is far superior to that of immunoassay-based detection [10, 40, 41].

The analysis of more complex proteins and peptides using MS instrumentation was for a long time restricted to research laboratories and biomarker discovery/validation workflows. This work has typically been low-throughput, using micro-flow or nano-flow LC separations over hours rather than minutes. However, recent work has focused on the development of higher throughput, more robust, quantitative protein/peptide analyses suitable for the rigours of clinical chemistry work. The MS analysis of proteins and peptides can be done either directly using the intact analyte(s) in a so-called ‘top-down’ approach, or in a ‘bottom-up’ approach following digestion with a protease such as trypsin, and by monitoring specific (tryptic) peptides as surrogate markers. The approach used depends on the target analyte; large proteins with m/z values exceeding the operating range of the MS instrument require proteolytic digestion. Further, some tryptic peptides offer better ionisation efficiency or better chromatographic peak shape than the intact species, and so the digestion step can be used to increase assay sensitivity. In the same manner as with quantitative small molecule MS analysis, isotopically labelled internal reference standards can be synthesised, either for whole peptides/proteins or proteolytic peptides, and matrix-based calibration standards can be produced, ideally from reference standards, such as the PTH1-84 reference standard mentioned earlier.

For low abundance proteins/peptides such as PTH1-84 (and PTH fragments) in serum, the complexity of the sample matrix necessitates thorough sample preparation prior to LC-MS analysis. For some small peptides, techniques traditionally used for small molecule analysis such as solid-phase extraction (SPE) may be useful, and have been successfully applied in a number of clinical assays, e.g., hepcidin [42] and insulin [43]. For larger proteins, or for those requiring more extensive analyte enrichment to allow detection at physiological concentrations, e.g., PTH [44], immunoaffinity extraction (sometimes referred to as immunocapture or immunoextraction) is a novel approach which is increasingly being used [45–48].

Matrix-assisted laser desorption ionisation coupled with time-of-flight MS (MALDI-TOF MS) has been widely used for peptide identification in proteomics studies. This technique has a wide m/z range, offers high-resolution MS, and a number of sophisticated de-convolution software packages are available to aid spectral interpretation. Furthermore, although MALDI-TOF cannot be directly connected to chromatographic separation techniques, these instruments can be used with immunoaffinity sample preparation and HPLC fraction collection methods to allow identification of low abundance proteins and peptides. Two studies using immunoaffinity coupled with MALDI-TOF MS to identify PTH fragments have been published. In 2006, Zhang et al. [10] immunoextracted PTH fragments from healthy controls, patients with chronic renal disease, and from healthy post-menopausal women given recombinant human PTH1-84. Extracted PTH fragments were analysed by: 1) MALDI-TOF MS following fractionation on a capillary LC system; and 2) directly by nano-LC-TOF-MS. Four PTH fragments (PTH34-84, PTH37-84, PTH38-84 and PTH45-84) were identified as the major circulating fragments in samples from renal patients and from healthy volunteers given PTH1-84 (at estimated concentrations from 10 to 100 pmol/L). All of these fragments, with the exception of PTH45-84, were also found in control samples. In 2010, Lopez et al. [49] carried out similar experiments, again using immunoaffinity to enrich the PTH fragments. However, this time the immunoaffinity process was automated by having the antibody (polyclonal goat anti-human PTH39-84) immobilised on a micro-columns embedded into pipette tips [mass spectrometric immunoassay (MSIA)]. In samples (n=12) from patients with renal failure, PTH fragments observed were consistent with those observed by Zhang et al. previously [10]. In addition, novel fragments with truncated N- and C-termini were also observed; these corresponded to PTH28-84, PTH48-84, PTH34-77, PTH37-77 and PTH38-77 (Figure 4). Lopez et al. also went on to develop a quantitative assay for these PTH fragments using a triple quadrupole mass spectrometer (see ‘Quantitative LC-MS/MS PTH assays’). It is especially interesting to note that in neither study were larger C-terminal PTH fragments observed, including the widely cited PTH7-84 variant to which the development of third-generation immunoassays can be largely credited.

Figure 4

Reconstructed MSIA MALDI-TOF MS spectra representative of samples from patients with renal failure (blue) and from healthy controls (red). Reproduced from Lopez et al. [49], with permission from Mary Lopez.

Quantitative LC-MS/MS PTH assays

Recently, two groups have published quantitative LC-MS/MS methods for the analysis of PTH1-84 in clinical samples [44, 49]. With these methods now available, it has been recognised that the development of a candidate reference method for PTH is now a possibility [16]. Both methods utilise immunoaffinity extraction with a C-terminal anti-PTH capture antibody, either immobilised onto polystyrene beads [44] or onto customised MSIA pipette tips [49]. In both methods, following washing steps to remove non-specific binding, the captured PTH1-84 and related fragments are digested using trypsin. Selected tryptic peptides are then quantified using LC-MS/MS. Absolute quantification is carried out by preparation of calibration standards using recombinant human PTH1-84. Kumar et al. [44] used isotopically labelled (¹⁵N) PTH1-84 as the internal reference standard, whereas Lopez et al. [49] used individual isotopically labelled tryptic peptides (one for each tryptic peptide monitored). Both groups used the quantification of the 1-13 amino acid tryptic peptide (SVSEIQLMHNLGK) as a surrogate for the measurement of PTH1-84. Both methods demonstrated good analytical performance, linearity and appropriate limits of quantitation (39.1 [44] and 16 pg/mL [49], respectively, from 1 mL sample). Kumar et al. also tested their assay for the 1-13 tryptic peptide for interference from other PTH fragments (PTH1-44, PTH7-84, PTH43-68, PTH52-84 and PTH64-84), PTHrP, and in haemolysed, lipaemic, and icteric samples. No significant interferences were identified.

In addition to the 1-13 peptide, Lopez et al. also included SRM transitions to monitor the presence of other tryptic peptides, specific to other PTH fragments. For example, the 7-13 tryptic peptide was used as a surrogate for PTH7-84. Echoing the findings using MALDI-TOF MS, the 7-13 tryptic peptide could not be detected (LLoQ 16 pg/mL) in the samples tested [49, 50]. We have since replicated this method in our laboratory using over 300 samples from patients with differing degrees of renal impairment [50–52].

Oxidised PTH

The discovery of the anabolic effects of PTH, and the subsequent use of recombinant PTH analogues as bio-therapeutic agents was an important development, and one which can be used to further our analytical ability to measure PTH variants in clinical samples. For these agents to be produced and licensed for use by regulatory authorities such as the FDA, extensive purity and drug stability testing is required. Much research is carried out to investigate the use of solvents and other excipient formulations to stabilise protein-based drugs. A number of studies using MS on the stability of PTH1-34 formulations have therefore been carried out. In addition, analytical methods to identify and characterise degradation products, plus in vitro studies to assess their potential biological activity have also been developed. The N-terminus of PTH1-84 contains a number of residues prone to chemical degradation. Amongst other possible degradation products such as deamidation of asparagine (ASN, N) residues, methionine (MET, M) oxidation is likely to be the most prevalent. In vitro studies using high-resolution MS to identify oxidation products, following exposure of these compounds to peroxides, have demonstrated that the methionine residues at positions 8 and 18 of PTH1-34 and PTH1-84 can become oxidised [38, 54]. Methionine 8 oxidises more slowly than methionine 18 [55], and both methionine residues can be singly or doubly oxidised (to sulphoxides and sulphones, respectively). It is also well established that all oxidised PTH variants are less biologically active than non-oxidised PTH [38, 56–60]. With these findings considered in the context of clinical PTH measurement, Hocher et al. [38, 61] raised a monoclonal antibody against oxidised PTH, and immobilised this antibody in microcolumns. High-resolution MS was used to demonstrate the efficiency of the antibody prior to use. These affinity columns were then used to remove oxidised PTH in clinical samples from haemodialysis patients prior to analysis of non-oxidised PTH species using immunoassay (second-generation, Roche PTH assay). It was demonstrated that a large, yet highly variable, proportion (>90% in some cases) of the total PTH in these samples was present in an oxidised form [38, 61].

Phosphorylated PTH

In our laboratory, we have employed a selected reaction monitoring (SRM) MSIA method similar to that of Lopez et al. [49]. The presence of phosphorylated PTH was looked for in clinical samples from patients with differing degrees of renal impairment. The immunoaffinity capture methodology and trypsin digestion steps were unchanged from the method described by Lopez et al., but additional SRM transitions were added to the MS method to allow the detection of the phosphorylated 14-20 tryptic peptide (HLNS_[phosphor] MER) and we used a custom-synthesised heavy isotope-labelled phosphorylated PTH14-20 internal reference standard. We were able to identify phosphorylated PTH in a number of clinical samples from patients with renal impairment and parathyroid cancer. In those samples where phosphorylated PTH was detected (n=58), a large range of phosphorylated PTH14-20 concentrations was observed [median (range) 52 (32–767 pg/mL)] [51, 52]. Further work is still required to determine the biological activity, or the significance of circulating phosphorylated PTH variants. An immunoassay for the detection of phosphorylated PTH has also been developed [62].