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Clinical Chemistry 54: 1317-1324, 2008. First published June 12, 2008; 10.1373/clinchem.2007.100644
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Right arrow Lipids, Lipoproteins, and Cardiovascular Risk Factors
(Clinical Chemistry. 2008;54:1317-1324.)
© 2008 American Association for Clinical Chemistry, Inc.

Lipids, Lipoproteins, and Cardiovascular Risk Factors

Potential of Sterol Analysis by Liquid Chromatography–Tandem Mass Spectrometry for the Prenatal Diagnosis of Smith-Lemli-Opitz Syndrome

William J. Griffiths1,a, Yuqin Wang1, Kersti Karu2, Emmanuel Samuel2, Shane McDonnell3, Martin Hornshaw3 and Cedric Shackleton4

1 Institute of Mass Spectrometry, School of Medicine, Swansea University, Swansea, UK; 2 The School of Pharmacy, University of London, London, UK; 3 Thermo Fisher Scientific, Hemel Hempstead, UK; 4 Children’s Hospital Oakland Research Institute, Oakland, CA.

aAddress correspondence to this author at: Institute of Mass Spectrometry, School of Medicine, Grove Building, Swansea University, Singleton Park, Swansea SA2 8PP, UK. Fax +44 (0)1792 295554; e-mail w.j.griffiths{at}swansea.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: Smith-Lemli-Opitz syndrome (SLOS), a severe disorder of cholesterol synthesis, is classically diagnosed prenatally by GC-MS analysis of sterols in amniotic fluid. Considering the current trend toward tandem mass spectrometry (MS/MS) methodologies, we developed prototype LC-MS/MS methods for accurate diagnosis of the disorder.

Methods: 3β-Hydroxysterols in amniotic fluid are oxidized with cholesterol oxidase to their corresponding 3-ketones, which are then derivatized with Girard P (GP) hydrazine in a "one-pot" reaction. The resulting GP-hydrazones give an improved response in electrospray (ES)–MS/MS owing to the presence of a charged quaternary nitrogen and are analyzed by reversed-phase LC-ES-MS/MS. Both capillary and conventional LC-MS/MS formats are suitable, and the method is also applicable to paper-absorbed blood spots.

Results: In a double-blind analysis of 18 amniotic fluid samples comprising 6 SLOS and 12 controls, the ratio of 7 + 8-dehydrocholesterol (7 + 8-DHC) to cholesterol was <0.02 [range 0.00–0.02, mean (SD) 0.01 (0.007)] in all control samples (intraassay variation 5.91%) and >0.20 [0.20–1.13, 0.79 (0.35)] in SLOS (intraassay variation 4.56%), corresponding to a difference in ratios between the 2 groups of at least a factor of 10. The limit of quantification was equivalent to that of 2 nL amniotic fluid injected on-column.

Conclusions: We describe a proof-of-concept for the prenatal diagnosis of SLOS. Further developments will be necessary to automate sample handling and reduce chromatographic time for the methodology to be used in pre- and postnatal diagnosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Smith-Lemli-Opitz syndrome (SLOS)1 is a serious disorder associated with dysmorphology and mental retardation. Because of the severity of the condition, development of diagnostic procedures has been critical. In 1993, gas chromatography–mass spectrometry (GC-MS) analysis of serum from a patient with SLOS showed an increased concentration of 7-dehydrocholesterol (7-DHC) (1). It was determined that this must be caused by defective 7-dehydrosterol-7-reductase, a final step in cholesterol synthesis, and GC-MS of serum cholesterol and 7-DHC became the accepted clinical method for SLOS diagnosis (2)(3). This method was further developed for prenatal diagnosis by measuring the sterols in amniotic fluid (3)(4)(5)(6)(7)(8)(9) or chorionic villus cells (10)(11). In addition to 7-DHC, serum and amniotic fluid contain 8-dehydrocholesterol (8-DHC), and this is included in some analyses (12). (For sterol structures, see the Supplemental Data that accompanies the online version of this article at http://www.clinchem.org/content/vol54/issue8.) GC-MS was also successful in retrospectively diagnosing SLOS from newborn blood spots, although this technique would be impractical for newborn screening (13). The drawback of GC-MS is the length of the chromatographic runs, and there have been several attempts to use other mass spectrometric techniques; however, the difficulty of ionizing these nonpolar sterols when conducting LC-MS has been a challenge. In an early study, Zimmerman et al. (14) evaluated TOF-MS for measurement of DHC and cholesterol in blood spots, with a goal of using this technique in newborn screening. In an even earlier study, Seedorf et al. (15) used particle beam LC-MS for diagnosing SLOS by plasma or amniotic fluid analysis. More recently, Johnson et al. (16) published a paper describing the use of a polar derivative in conjunction with electrospray (ES)–tandem mass spectrometry (MS/MS) for SLOS screening. In 2007, Pitt (17) reported the monitoring of a sulfated ketopregnadienediol metabolite in urine by ES-MS/MS for SLOS screening.

We previously developed a methodology for the analysis of sterols and oxysterols by liquid chromatography (LC)–ES-MS/MS and LC-ES-MSn. In this method, sterols are first oxidized by cholesterol oxidase, replacing the 3β-hydroxy-5-ene group with the 3-oxo-4-ene moiety (18)(19), then derivatized with Girard P (GP) reagent, forming a derivative with excellent ES-MS/MS properties (Fig. 1 ). Here we demonstrate the use of this method for the accurate prenatal diagnosis of SLOS through amniotic fluid analysis.


Figure 1
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  		Figure 1. Oxidation/derivatization of cholesterol and 7-DHC.

Although oxidized/derivatized 7-DHC is drawn as the 4,7-diene, isomerization to the 4,6-diene is plausible.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
materials
We purchased sterol standards, GP reagent [1-(carboxymethyl)pyridinium chloride hydrazide], and cholesterol oxidase from Streptomyces sp. from Sigma-Aldrich Ltd. and Sep-Pak C18 from Waters Corp. Amniotic fluid was provided by the Kennedy Krieger Institute, Johns Hopkins University, with Institutional Review Board approval. Blood spots from SLOS-affected patients were from whole venous blood spotted onto standard newborn screening cards and provided by the Heritable Disorders Branch, National Institute of Child Health and Human Development, with institutional approval. These blood spots were intended to emulate newborn screening from Guthrie card samples, but were actually samples from older infants and children.

derivatization
We added 10 µL amniotic fluid to 2.5 µL cholesterol oxidase from Streptomyces sp. (2 g/L, 44 U/mg protein) in 1 mL buffer (50 mmol/L KH2PO4, pH 7) and incubated the mixture at 37 °C for 60 min. The reaction was stopped with 2 mL methanol, and we added 150 mg GP hydrazine and 150 µL glacial acetic acid (Fig. 1Up ) and left the mixture at room temperature overnight. We separated the steroid GP hydrazones from excess reagent by extraction on a Sep-Pak C18 column (1 by 0.8 cm) using a recycling method. We applied the GP reaction mixture (approximately 3 mL of 70% methanol) directly to a Sep-Pak C18 column followed by 1 mL of 70% methanol and 1 mL of 35% methanol. The combined effluent (5 mL) was diluted with 4 mL of water. The resulting mixture (9 mL in 35% methanol) was again applied to the column followed by a wash with 1 mL of 17% methanol. We added 9 mL water to the combined effluent, producing a 19-mL sample in 17.5% methanol. This was again applied to the column, followed by a wash with 10 mL of 10% methanol. The extracted GP derivatives were then eluted with 2 portions of 1 mL methanol followed by 1 mL chloroform/methanol (1:1, vol/vol). The chloroform/methanol fraction was dried under a gentle stream of N2 and reconstituted in 1 mL methanol, and we combined the 3 fractions.

For blood spot analysis, a 10-mm-diameter sample was punched from the filter paper and sliced into small pieces. We added 100 µL isopropanol followed by 1 mL buffer (50 mmol/L KH2PO4, pH 7) containing 2.5 µL cholesterol oxidase from Streptomyces sp. (2 g/L, 44 U/mg protein). The mixture was incubated at 37 °C for 60 min and processed further as above.

lc-ms/ms
In this proof-of-principle study, we evaluated both capillary and conventional LC-ES-MS/MS methods. Capillary LC-ES-MS/MS was performed on both an LCQ ion-trap mass spectrometer (Thermo Fisher Scientific) and a quadrupole-TOF instrument (Q-TOF; Waters), whereas conventional LC-ES-MS/MS was performed on a TSQ Quantum Access (Thermo Fisher Scientific) triple quadrupole mass spectrometer.

For capillary-LC-MS2 analysis, we diluted 6 µL of the combined methanol fractions (equivalent to 20 nL amniotic fluid) with 4 µL of 0.25% formic acid and injected 1–5 µL (equivalent to 0.002–0.01 µL of amniotic fluid) onto either a PepMap C18 column (180 µm by 150 mm, 3 µm, 100Å; Dionex) or a Hypersil Gold C18 column (180 µm by 100 mm, 3 µm; Thermo Fisher) using a WPS 3000 well plate autosampler, part of an Ultimate 3000 Capillary HPLC system (Dionex). Mobile phase A was 0.1% formic acid in 50% methanol; mobile phase B was 0.1% formic acid in 95% methanol; and transport solvent was 0.1% formic acid in 60% methanol. The gradient program started at 30% B, which was maintained for 6 min, increasing to 73% B over 4 min, then to 77% B over a further 2.5 min, and finally to 80% B over 2.5 min. The mobile phase composition was maintained at 80% B for a further 10 min before returning to 30% in 6 s. The column was reconditioned with this mobile phase composition for 24 min, 54 s. The total run time was 50 min, and the flow rate 0.8 µL/min. For the initial identification of sterols in amniotic fluid, MS, MS2 ([M]+->), and MS3 ([M]+->[M-79]+->) spectra were recorded on the LCQ ion-trap (Fig. 2 ). For the acquisition of both MS2 and MS3 spectra, we set the precursor ion isolation width at 2 and the collision energy at 45%. The collision gas was helium. For the quantification of sterols, we recorded only MS2 spectra and generated reconstructed ion chromatograms (RICs) for the [M]+-> [M-79]+ transitions over the duration of the LC-MS2 run. Capillary LC-MS/MS was also performed on the Q-TOF mass spectrometer using a shorter run time, the details of which are provided in the online Data Supplement.


Figure 2
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  		Figure 2. MS2 and MS3 pathways applicable to fragmentation of oxidized/derivatized cholesterol (A) and 7-DHC (B).

Although oxidized/derivatized 7-DHC is represented as the 4,7-diene, isomerization to the 4,6-diene is plausible. On collisional activation, the 4,7-diene can isomerize to the 4,6-diene. The MS/MS spectra recorded on Q-TOF and TSQ Quantum Access are a composite of MS2 and MS3 pathways.

We analyzed sterols in blood spots by nano-ES-MSn on the LCQ ion-trap following oxidation/derivatization where 1 µL of the combined methanol fractions (1/3000 of the blood spot) was loaded into a metal-coated nanospray capillary and electrosprayed into the ion-source. MS, MS2, and MS3 spectra were recorded as described above.

We performed conventional LC-MS/MS analysis using a Surveyor MS pump (Thermo Fisher Scientific) and TSQ Quantum Access triple quadrupole mass spectrometer. We injected 5 µL sample prepared as described above (equivalent to 0.01 µL amniotic fluid) via a Micro AS (Thermo Fisher Scientific) autosampler onto a Hypersil Gold C18 column (50 by 2.1 mm, 1.9 µm). Mobile phase A was 50% methanol, and mobile phase B was methanol/isopropanol/water (75/20/5, vol/vol/vol), both containing 0.1% formic acid. The flow rate was 400 µL/min. The gradient program started at 50% B, rising to 100% B in 1 min, maintained at 100% B for 2 min before returning to 50% B in 6 s, and reequilibrating at 50% B for a further 1 min, 54 s, giving a total run time of 5 min. Quantification was performed using the following multiple reaction monitoring (MRM) transitions, with collision energies given in parenthesis: m/z 518.4->439.4 (28 eV) + 163.1 (40 eV) or 518.4->163.1 (40 eV), and 516.4->437.4 (24 eV) + 137.1 (40 eV) or 516.4->137.1 (40 eV) (see Fig. 2Up ). The collision gas was argon.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
We have previously demonstrated that a strategy of sterol oxidation with cholesterol oxidase followed by derivatization of the resulting 3-oxo group with GP hydrazine (Fig. 1Up ) enhances ES ion current by 2 to 3 orders of magnitude (18)(19). Furthermore, derivatization with GP hydrazine, which introduces a positively charged quaternary nitrogen to the molecule, enhances solubility in aqueous solvents and thus increases the diversity of mobile phase available for reversed-phase LC separations. The oxidation/derivatization reactions can be performed on free sterols from µL quantities of amniotic fluid without any prior saponification of esters. The reactions proceed efficiently for both the 3β-hydroxy-5-ene and 3β-hydroxy-5,7-diene sterols giving 3-GP hydrazones of the 3-oxo sterols (18)(19). Although the derivatization reaction is performed overnight, the whole procedure of oxidation, derivatization, and extraction requires little manual effort and theoretically could be adapted to a robotic liquid handling devise to automate the procedure. Further, the limit of quantification of the analytical method (equivalent of 0.002–0.01 µL amniotic fluid injected on-column) is such that sample quantities can be greatly reduced and the method miniaturized (20). Preliminary data indicates that the methodology is equally applicable to sterols in blood spots on filter paper (as representative of Guthrie cards).

A major feature of much of our work is the use of capillary LC for the analysis of sterols (18). Thus, we initially evaluated the use of capillary LC combined with MSn for the analysis of cholesterol and DHC in amniotic fluid. Our goal was simply to measure the ratio of DHC to cholesterol in control samples and those from SLOS-affected pregnancies, and to evaluate if this ratio could be used to allow the accurate prenatal diagnosis of SLOS. Although sample availability is not generally an issue regarding amniotic fluid, the limit of quantification of the capillary-LC-MSn system allows volumes as little as 0.002 µL of amniotic fluid to be injected on column (i.e., containing much less than 1 ng sterol (9)). Cholesterol is the major sterol in control amniotic fluid (10–100 mg/L after saponification (9)), and following oxidation/derivatization gives a [M]+ ion at m/z 518.4 (Fig. 1Up ). Although resolved from cholesterol, oxidized/derivatized 7-DHC was not resolved from its isomer 8-DHC on the capillary column, so it generated a composite peak (see the online Data Supplement). Oxidized/derivatized 7-DHC and 8-DHC both give an [M]+ ion of m/z 516.4, as does desmosterol (3β-hydroxycholest-5,24-diene), another component of amniotic fluid; desmosterol elutes earlier than 7 + 8-DHC, however. As well as being chromatographically separated, oxidized/derivatized cholesterol gives different fragment ions in its MS2 and MS3 spectra than those generated by 7/8-DHC or desmosterol (Fig. 2Up and the online Data Supplement). Unfortunately, an authentic standard of 8-DHC is not commercially available, but we expect it would provide a mass spectrometric response similar to that of 7-DHC in the ion-trap mass spectrometer. This allows the MS2 transitions 518->439 and 516->437 to be monitored for the determination of the relative abundance of cholesterol and 7 + 8-DHC. We generated a 5-point standard curve for the relative quantification of 7-DHC to cholesterol using authentic standards for molar ratios between 0.02 and 1 over the range of 0.6–30 pg on-column, giving an R2 of 0.99, indicating that the derivatives are suitable for the quantification of 7-DHC and cholesterol and by inference 7 + 8-DHC and cholesterol. Using capillary-LC-MS2 with the LCQ ion-trap, the detection limit for the oxidized/derivatized steroids was 1 pg on-column.

In a double-blind analysis of 18 amniotic fluid samples comprising 6 SLOS and 12 controls, the DHC-to-cholesterol ratio was <0.02 [range 0.00–0.02, mean (SD) 0.01 (0.007)] in all control samples (intraassay variation 5.91%) and >0.20 [0.20–1.13, 0.79 (0.35)] in SLOS (intraassay variation 4.56%), corresponding to a difference in ratios between the 2 groups of at least a factor of 10 (Fig. 3 , Table 1 ). The interassay variation was 14%. Direct comparison of concentrations determined here with those made some years earlier by GC-MS is not viable because some samples have been stored frozen for a number of years since GC-MS analysis, and no assessment was made of sample degradation. This did not detract from the efficacy of the method in diagnosing SLOS. Although the above experiment provided a correct diagnosis, the chromatographic run time of 50 min, plus the necessity of running at least 1 blank between biological samples, would not meet the need for high-throughput prenatal analysis, much less newborn screening.


Figure 3
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  		Figure 3. Analysis of oxidized/derivatized cholesterol and 7 + 8-DHC from control (sample 34090) (A) and SLOS-affected (sample 31576) (B) amniotic fluid by capillary LC-MS2 on the ion-trap mass spectrometer.

Upper panels show RICs for the MS2 transition 518->439 (cholesterol), and lower panels RICs for the MS2 transition 516->437 (7 + 8-DHC). Although the absolute retention time of a given sterol varied on different days, the order of elution between 7 + 8-DHC and cholesterol did not change.


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  		Table 1. Ratio of DHC to cholesterol in amniotic fluid from SLOS-affected and nonaffected pregnancies.1

Although capillary LC combined with low-flow-rate-ES provides maximum performance in terms of theoretical chromatographic resolution and ionization efficiency (21), the severe restriction of run time makes it impractical for high-throughput screening such as in newborn blood spot analysis. We thus turned our attention to conventional liquid chromatography. Using a 50 by 2.1 mm C18 column and a water/methanol/isopropanol mobile phase flowing at 400 µL/min, oxidized/derivatized DHC and cholesterol could be separated from the solvent front and eluted in 3 min, giving a total run time of 5 min. Despite no longer being chromatographically separated, DHC and cholesterol give different MRM transitions, allowing their deconvolution (Fig. 2Up ). Using the combined transitions 516.4->437.4 + 137.1 and 518.4->439.4 + 163.1, the ratio of 7 + 8-DHC to cholesterol in control samples (range 0.00–0.11) was always found to be at least 8 times lower than that in the SLOS samples (data not shown). As desmosterol will coelute with 7 + 8-DHC and also give a 516.4->437.4 transition, and the second 13C isotopic peak of 7 + 8-DHC will also give the transition 518.4->439.4, it was decided to exclude these transitions. The 516.4->137.1 and 518.4->163.1 transitions, although giving less ion current (detection limit 5 pg on-column), may provide a better measure of the DHC-to-cholesterol ratio (Fig. 4 ). Using these transitions, the ratio was found to be 0.01 or below [0.00–0.01, 0.003 (0.004)] for the control samples (intraassay variation 15.4%) and at least 38 times higher [0.38–2.32, 1.29 (0.81)] for the SLOS samples (intraassay variation 9.7%, interassay variation 32%). Table 1Up compares the current LC-MSn and LC-MS/MS results with those obtained in earlier studies by GC-MS following saponification and silation. (Again, the samples were analyzed by GC-MS a number of years before the current LC-MS study.)


Figure 4
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  		Figure 4. Analysis of oxidized/derivatized cholesterol and 7 + 8-DHC from control (sample 34106) (A) and SLOS-affected (sample 31576) (B) amniotic fluid by conventional LC-MS/MS using MRM on a triple quadrupole mass spectrometer.

Upper panels show RICs for the MRM transition 518.4->163.1 (cholesterol), and lower panels RICs for the MRM transition 516.4->137.1 (7 + 8-DHC).

In this report, we present a potential method for the accurate identification of SLOS from amniotic fluid samples. The method requires only 10 µL amniotic fluid for oxidation/derivatization, of which only 0.01 µL or less is injected on the LC-MS system. By using capillary LC-MSn, it is possible to chromatographically separate 7 + 8-DHC from desmosterol and cholesterol and perform relative quantification using the transitions 516->437 for 7 + 8-DHC and 518->439 for cholesterol (Fig. 3Up ). Viable clinical methods must offer some advantages over existing methodologies (in this case GC-MS), and in our preliminary study this was not achieved. Capillary-LC-MSn still required long run times and derivatization. It is likely that derivatization will always be necessary because of the nonpolar nature of the analytes, but chromatography times can certainly be shortened, and automated sample processing is being developed. In an effort to make the method more compatible with clinical screening, the original chromatography was scaled up to a conventional format using a 2.1 by 50 mm column and a flow rate of 400 µL/min. Fast chromatography was achieved, with runs being completed in 3 min. Although chromatographic peak shape and separation becomes compromised, the use of the more specific 516.4->137.1 and 518.4->163.1 transitions allows the accurate identification of SLOS.

A drawback of the method is that separate analysis of both 7- and 8-DHC has not been achieved. Our results for 7 + 8-DHC quantification are based on the assumption that oxidized/derivatized 7-DHC and 8-DHC behave identically in the mass spectrometer. This is likely to be true in the MS2 event in the ion-trap, where only 1 major fragmentation pathway is followed (see the online Data Supplement), but when MS/MS is performed on both the tandem quadrupole and Q-TOF instruments, where fragmentation is dispersed over a wide range of fragment ions, it is to be expected that the 2 isomers will give different relative abundance for each fragment ion. This difference provides the explanation for the discrepancy in ratios of 7 + 8-DHC to cholesterol determined on the ion-trap and quadrupole instruments. Nevertheless, both instruments allow the accurate identification of SLOS.

The instability of the ring B unsaturated sterols remains a problem as in other methodologies, maybe more so in the method described here because the DHCs are unstable in both the free and derivatized form. Much of the autoxidation can be prevented by protecting samples from light and completing the extraction and derivatization rapidly. The derivatives used for GC-MS effectively protect the compounds from further degradation, but we have found that oxidized/derivatized cholesterol and 7 + 8-DHC required for LC-MS analysis cannot be stored in aqueous solvents for extended periods of time. This problem is overcome by diluting the sample into aqueous solvents immediately before injection on to the LC column. We recommend that samples not be stored in aqueous solvents for periods in excess of 4 h.

We are not presenting a turnkey LC-MS/MS method for the prenatal and neonatal diagnosis of SLOS, but rather results of preliminary investigations toward such a method. It is clear that in almost all instances there is an inexorable move from GC-MS to LC-MS/MS, necessary to meet the requirements of automated sample handling (21). In newborn screening using blood spots, which may be introduced in the future for SLOS diagnosis, only LC-MS/MS would be viable. As a prelude to a detailed blood spot study, we have tested our oxidation/derivatization chemistry and ES-MSn analysis on a trial set of 6 blood spots from SLOS patients. The results are presented as Supplemental Data, which shows ES-MS, -MS2, and -MS3 spectra that confirm the increased abundance of 7 + 8-DHC in the blood from an SLOS patient.

The next requirement is improvement in the chromatography. The use of ultra–small particle size (<1.7 µm) chromatography may allow rapid (<5 min run times) and specific measurement of cholesterol and the DHCs. What we have demonstrated here is a suitable derivatization technique for nonpolar sterols and the MS/MS fragmentations needed for specific analysis of cholesterols and the DHCs. The proof of the concept lay in the correct diagnosis of SLOS in patient samples.


   Acknowledgments
 
Grant/Funding Support: This work was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC grants BB/C515771/1 and BB/C511356/1) and Swansea University. C. Shackleton is supported by the NICHD branch of the US National Institutes of Health (grant HD053036).

Financial Disclosure: None declared.

Acknowledgments: We are grateful for the provision of deidentified amniotic fluid samples from Kennedy Krieger Biochemical Genetics Laboratory (courtesy of Richard Kelley and Lisa Kratz) and blood spots from the Heritable Disorders Branch, National Institute of Child Health and Human Development, Bethesda, MD (courtesy of Forbes D. Porter).


   Footnotes
 
1 Nonstandard abbreviations: SLOS, Smith-Lemli-Opitz syndrome; GC-MS, gas chromatography–mass spectrometry; DHC, dehydrocholesterol; ES, electrospray; MS/MS, tandem MS/MS; GP, Girard P; LC, liquid chromatography; MS2, [M]+->; MS3, [M]+->[M-79]+->; RIC, reconstructed ion chromatogram; MRM, multiple reaction monitoring.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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