Use of Stable Labeled Internal Standards LC-MS/MS Method for the Detection and Confirmation of Inherited Metabolic Diseases in Sick Infants

 

Mohammed Abdel-Hamid and Leyla H. Sharaf*

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, Jabriya, Kuwait, PO Box 24923, Safat 13110 Kuwait.

*Corresponding Author E-mail: ls20504@hsc.edu.kw

ABSTRACT:

The objective of this study was to indicate the potential of the stable labeled internal standards liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, as a diagnostic method for the detection and confirmation of inherited metabolic diseases (IMDs) in sick infants.

 

A total of 2050 dried blood spot samples of sick infants with suspected IMDs were routinely analyzed by tandem mass spectrometry (MS/MS). Samples showing positive results of particular IMDs were further measured by LC-MS/MS using labeled internal standards to confirm the diagnosis and to determine accurately the concentrations of the diagnostic biomarkers in the provided samples.

 

Labeled internal standards LC-MS/MS method has confirmed the diagnosis of IMDs in 19 symptomatic infants with metabolic diseases namely;  malonic aciduria (MA) (1 case), methylmalonic aciduria (MMA) (6 cases), ethylmalonic aciduria (EMA) (4 cases), Canavan’s disease (CD) (1 case), Ornithine carbamoyltransferase deficiency (OCTD) (1 case), classical galactosemia (GAL) (3 cases), homocystinuria (HCY) (2 cases) and Zellweger Syndrome (ZS) (1 case). The above disorders were not appropriately detected by routine MS/MS screening. The concentrations of the diagnostic biomarkers of these disorders were accurately measured and were found to be markedly elevated compared to control samples. 

 

The obtained results proved that the described labeled internal standards LC-MS/MS method is a powerful and complementary diagnostic technique to routine MS/MS. The method permits specific detection and quantitation of the diagnostic biomarkers of IMDs in sick infants. The technique is simple and reliable for a rapid differential diagnosis of the metabolic disorders and can be tailored according to physician’s requests.

 

 

INTRODUCTION:

Inherited metabolic diseases (IMDs) are mainly a group of inherited autosomal recessive disorders which may carry serious consequences to the affected newborns and infants such as mental retardation, physical handicaps or even fatality. The prevalence of IMDs is relatively high among Kuwaiti population due to consanguinity¹. For diagnostic purposes, accurate and rapid diagnosis of the suspected metabolic disorders is important to identify the metabolic disease and to start early management and control. Several analytical techniques such as HPLC ², GC/MS ³, electrophoresis ⁴ and immunoassays ⁵ were used in clinical biochemical laboratories for the diagnosis of IMDs.

 

Recently, tandem mass spectrometry (MS/MS) has been used extensively for metabolic screening ^6-8. The application of MS/MS has significantly improved the metabolic screening process by increasing the spectrum of metabolic diseases to be diagnosed and by increasing the number of cases to be detected. MS/MS permits metabolic screening of more than 30 IMDs in a single dried blood spot in less than 3 minutes and allows more than 500 samples to be analyzed in few hours due to high sample throughput ⁹.  Amino acid disorders, organic acid disorders, urea cycle disorders and fatty acid oxidation defects are among the most common IMDs detected by MS/MS technology ⁹.

 

Although MS/MS screening has improved the screening process, however the technique has some limitations e.g. lengthy extraction and derivatization process, limited diagnostic biomarkers to be detected, inaccurate measurements of the diagnostic biomarkers in patient samples, inability to detect serious metabolic disorders e.g. ethylmalonic aciduria and inability to differentiate metabolic disorders (methylmalonic and propionic acid disorders) or biomarkers (leucine and isoleucine amino acids) for  MSUD. Recently, LC-MS/MS procedures have been used in specialized biochemical laboratories for the diagnosis of some IMDs which could not be screened by routine MS/MS ^10-16. We have introduced the metabolic screening service using tandem mass spectrometry at our institution for the diagnosis of inherited metabolic diseases in sick infants, in collaboration with the Government Pediatrics and Maternity Hospitals ¹⁷. The Guthrie cards containing the dried blood spots (DBS) of sick infants were sent to our laboratory for analysis by MS/MS ¹⁷. Our work was further extended by introducing the labeled internal standards LC-MS/MS to our screening program to broaden the scope of the metabolic diagnosis and to permit accurate measurements of the diagnostic biomarkers in different patient samples such as DBS or urine or plasma to follow up the progress of the treatment process. This presentation reports on the utility of the labeled internal standards LC-MS/MS method as an accurate and confirmatory method for the diagnosis of some specific disorders, where routine MS/MS screening was not appropriate.

 

MATERIALS AND METHODS:

Materials Labeled internal standards of d₃-methylmalonic acid (d₃-MMA), d₂-orotic acid (d₂-ORT), d₅-phenylalanine and d₆-galactose were obtained from Cambridge Isotope Laboratories (Andover, MA, USA). Plasma and urine samples were stored at -30^oC until analysis and the dried blood spots (DBS) cards were kept in specimen bags at 4^oC.

 

Instrument and conditions:

LC-MS/MS analyses were performed using Micromass Quattro LC bench-top triple quadrupole instrument interfaced with a Z-spray electrospray ionization (ESI) source which may be operated in either positive or negative mode. The mass spectrometer was connected to Waters 2695 Separation Module and autosampler (Waters). All operation and acquisition processes were controlled by Masslynx software of the instrument. The tuning parameters of the mass spectrometer were adjusted and optimized (Table 1). Sample analysis by LC-MS/MS was performed on MS XTerra^® RP-C8 (150 mm x 4.6 mm; 5µm) column. Two mobile phases were used; (A) of acetonitrile /ammonium acetate, 10 mM (50:50 v/v) at flow rate 400 µl/min for positive ESI and (B) of acetonitrile/water/formic acid (80:20:0.05 v/v/v) at flow rate 400 µl/min for negative ESI. Quantitative analysis was performed using Multiple Reaction Monitoring (MRM) scanning mode (Table 1).

 

Analysis of Patients’ Samples :

LC-MS/MS analysis of malonic aciduria (MA), methylmalonic aciduria (MMA), ethylmalonic aciduria (EMA), and Canavan’s disease (CD) samples: A 50 µl aliquot of urine sample of sick infant, previously filtered using 0.45 µm Millipore membrane filter, was transferred into an HPLC-vial, diluted to 980 µl with water and then mixed with 20 µl aliquot of d₃-MMA (100 ng/µl) in methanol/water (1:9). A 20 µl aliquot of the sample was injected into the LC-MS/MS and analyzed using negative ESI-MRM scanning (Table 1).

 

LC-MS/MS analysis of ornithine carbomyltransferase deficiency (OCTD) samples: A 50 µl aliquot of  urine sample of a sick infant, previously filtered using 0.45 µm Millipore membrane filter, was transferred into an HPLC- vial, diluted to 980 µl with water and then mixed with 20 µl aliquot of d₂-orotic acid (100 ng/µl) in methanol/water (1:9). A 20 µl aliquot of the sample was injected into the LC-MS/MS and analyzed using negative ESI-MRM scanning (Table 1).

 

LC-MS/MS analysis of samples of classical galactosemia: The Guthrie card containing the dried blood spots (DBS) of a sick infant was punched and a blood spot (~3 mm) was transferred into a capped 10-ml glass tube and  extracted by shaking with 100 µl of acetonitrile/ammonium acetate solution, 10mM (1:1) and 100 µl (1 µg/µl) of d₆-galactose in ammonium acetate solution, 10 mM for 10 min. A 20 µl aliquot of the solution was directly injected into the LC-MS/MS using negative ESI-MRM scanning (Table 1).

 

LC-MS/MS Analysis of samples of homocystinuria (HCY) or peroxisomal diseases (Zellweger Syndrome)(ZS):A 50 µl aliquot of plasma sample of a sick infant was transferred into a 1.5-ml  Eppendorff tube, mixed with 10 µl aliquot of d₅-phenylalanine solution (1ng/µl) in water/methanol (9:1) and 200 µl of acetonitrile as a protein precipitant. The sample was vortexed for 1 min and then centrifuged at 13000 rpm for 10 min. The clear supernatant was separated and the solvent was evaporated under N₂ at 80^oC. The residue was reconstituted in 80 µl of methanol/water (50:50) and a 20 µl aliquot was injected into the LC-MS/MS using positive ESI-MRM scanning (Table 1).

 

Calibration curve:

 Stock solutions of the diagnostic biomarkers (malonic acid, methylmalonic acid, ethylmalonic acid, orotic acid, N-acetylaspartic acid, galactose-1-P, homocysteine and L-pipecolic acid) and the labeled internal standards (d₃-methylmalonic acid, d₂-orotic acid, d₆-galactose and d₅-phenylalanine) were separately prepared in methanol at  concentrations 1µg/µl and stored at 4^oC. Working solutions of the diagnostic biomarkers were freshly prepared in water/methanol (9:1) at appropriate concentrations. Calibrators were separately prepared in urine or blood or plasma at concentrations specified in Table 2. The calibrators were similarly treated as shown under “analysis of patients’ samples”.

 

 

Table No. 1: Tuning parameters for mass spectrometric analysis of the diagnostic biomarkers and the labeled internal standards

Malonic acid                                    Negative                        15                                   11                                   102.9>59.1

Methylmalonic acid                        Negative                        15                                   11                                   117.2>73.3

d₃-Methylmalonic                           Negative                        15                                   11                                   120.2>76.3

Ethylmalonic acid                           Negative                        15                                   11                                   130.8>87.3

N-Acetylaspartic acid                     Negative                        15                                   11                                   174.2>88.2

Orotic acid                                                            Negative                        15                                   11                                   155.2>111.3

d₂-Orotic acid                                   Negative                        15                                   11                                   157.3>113.2

Gal-1-P                                              Negative                        22                                   15                                   259.2>79.2

d₆-Galactose                                     Negative                        22                                   15                                   185.2>92.2

L-Pipecolic acid                               Positive                          22                                   15                                   130.1>84.2

Homocysteine                                  Positive                          22                                   15                                   136.1>90.1

*Source Temperature: 100^oC, Desolvation Temperature: 250^oC

 

Table No. 2: Calibrations of the diagnostic biomarkers in biological matrixes as determined by the labeled internal standards LC-MS/MS method

Diagnostic biomarker	Concentration range	Linear regression equation (y = a + bx)***	Correlation coefficient (r)
Malonic acid*	250-1000 µmol/l	y = -0.075 + 0.002 x	0.9981
Methylmalonic acid*	250-1000 µmol/l	y = 0.098 + 0.015 x	0.9983
Ethylmalonic acid*	250-1000 µmol/l	y = 0.021 + 0.002 x	0.9984
Orotic acid*	250-1000 µmol/l	y = 0.061 + 0.003 x	0.9911
Gal-1-P**	0.08-3.85 mmole/l	y = 0.010 + 0.035 x	0.9925
N-acetylaspartic acid*	250-1000 µmol/l	y = 0.085 + 0.003 x	0.9977
Homocysteine#	30-90 µmol/l	y = 1.18 + 0.008 x	0.9900
L-pipecolic acid#	30-90 µmol/l	y = 7.240 + 0.220 x	0.9912

Calibration curves were prepared in: Urine*, DBS**, Plasma#  ***Linear regression equation , y= PA₁/PA₂, x= C µmol/l,   PA₁=Peak Area (diagnostic biomarker), PA₂= Peak Area (labeled IS)

 

Validation and Quantification:

Validation parameters such as linearity, limit of quantification (LOQ), precision and recovery percentages were established for each diagnostic biomarker. The concentration of the diagnostic biomarker in  patient’s sample (urine or DBS or plasma) was calculated from the linear regression equation representing the calibration curve (Table 2).

 

RESULTS:

LC-MS/MS procedures for measurement of selected diagnostic biomarkers of particular IMDs have been developed (Table 1).

 

 

The mass spectrometer tuning parameters and chromatographic conditions were optimized to achieve good detection and resolution of the analytes. The diagnostic biomarkers; malonic acid, methylmalonic acid, ethylmalonic acid, orotic acid, N-acetylaspartic acid and Gal-1P; were measured by negative electrospray LC-MS/MS method, whereas homocysteine and L-pipecolic acid were measured by positive electrospray LC-MS/MS procedure.  Figure 1 showed the MRM chromatograms of a urine sample spiked with a mixture of malonic, methylmalonic, ethylmalonic acids and d₃-methylmalonic acid as a labeled IS.

 

 

Fig.1: Negative electrospray LC-MS/MS chromatograms of  urine sample spiked with Ethylmalonic acid ¹, d₃-Methylmalonic acid ², Methylmalonic acid ³, and Malonic acid ⁴.

 

 

 

Unlike, tandem mass spectrometry or gas chromatography/mass spectrometry (GC/MS), simple preliminary methods were used to prepare patient’s samples before analysis by LC-MS/MS e.g. dilute and shoot for urine samples (MA, MMA, EMA and CD), protein precipitation for plasma samples (HCY, ZS) and direct extraction with mobile phase for DBS samples (Galactosemia). For quantitative measurements, the calibration curves for the diagnostic biomarkers in the specified biological media were initially established from by plotting the peak area ratios of the MRM–chromatograms of biomarker and IS vs. concentration of biomarker. The selected concentration ranges of calibrators, linear regression equations with regression coefficients and LOQ values were listed in Table 2. The % relative standard deviations (RSD%) for the inter-day, intra-day precision were found to be less than 20.5% and the recovery percentages for inter-day and intra-day accuracy were more than 83.7%. The calculated concentrations of the biomarkers for the diagnosed cases were reported in Table 3.

 

As shown, a rare case of MA was confirmed by the presence  of malonic and methylmalonic acids in urine at concentrations of 265 and 350 µmol/l, respectively. MMA patients were identified by the presence of methylmalonic acid in urine at concentration up to 3520 µmol/l ¹⁵.  EMA patients were diagnosed by the detection of a high concentration of ethylmalonic acid in patient’s urine up to 663 µmol/l ¹⁸.  Metabolic screening of the urine samples by GC/MS indicated the presence of malonic, methylmalonic and ethylmalonic acids, respectively, whereas metabolic profiling of urine samples by MS/MS indicated the presence of  malonylcarnitine, propionylcarnitine and butyrylcarnitine at m/z 360, 274, and 288, respectively (Figure 2 A, B, C). 

 

Furthermore, a case of OCTD was also identified and confirmed by the presence of orotic acid in patient’s urine at a concentration of 1405 µmol/l (Figure 3) ¹¹

 

 

A

B-        

C-

Fig.2: Positive electrospray MS/MS profiles of urine samples of sick infants with malonic aciduria (A), methylmalonic aciduria (B) and ethylmalonic aciduria (C).

 

Fig. 3: Negative electrospray LC-MS/MS chromatograms of urine sample of  sick infant with OCTD. Orotic acid ¹ and d₂-Orotic acid ².

Three cases of classical galactosemia were identified by high concentration of Gal-1P in DBS at 2.5-3.5 mmol/l ¹³. The elevated concentration of N-acetylaspartic acid in patient’s urine at a concentration of 1438 µmol/l proved a case of CD ¹⁰. The high plasma concentrations of homocysteine at 31 and 52 µmol/l confirm cases of homocystinuria ¹² and a high  plasma level of L-pipecolic acid at 53.5 µmol/l confirms  peroxisomal disorders of Zellweger Syndrome ¹⁴.

 

DISCUSSION:

Although tandem mass technology has revolutionized the screening process of IMDs in DBS, however the technique

is still inappropriate for the diagnosis of some serious organic acid disorders as the characteristic mass ions of the diagnostic biomarkers may be either missed (MA) or not  detected (EMA) in DBS. Furthermore, the metabolic disorders; methylmalonic and propionic acid disorders  (MMA / PPA);  could not be differentiated because the diagnostic biomarker (propionylcarnitine) is the same for both disorders.  Moreover, serious metabolic disorders such as OCTD, CD, classical galactosemia and peroxisomal disorders could not be detected during routine tandem mass screening. Therefore, diagnosis by tandem mass alone does not provide a comprehensive solution for the diagnosis of all metabolic disorders and normal profiles should not be taken to rule out  IMDs. Although, GC/MS is  useful for the diagnosis of IMDs ³, particularly organic acid disorders, however the technique is time-consuming (run time ≤ 1 h), and tedious as it involves multiple extraction and derivatization processes.  In this respect, LC-MS/MS may provide a good solution for the diagnostic and confirmatory purposes. Using labeled internal standards LC-MS/MS, a very rare case of MA was detected and the organic acid disorders (MMA and PPA) were differentiated. The LC-MS/MS method for detection of ethylmalonic acid permits diagnosis of serious organic acid disorder of EMA in urine samples. The high concentrations of Gal-1P in DBS samples confirms the diagnosis of classical galactosemia in patients with severe hepatic failure. The results were in good agreement with the reported enzymatic method¹⁹. The elevated concentration of orotic acid in urine confirms a rare case of OCTD. Orotic acid was the only diagnostic biomarker for the diagnosis of OCTD. The test was also useful for the diagnosis of patients with hyperammonemia and other urea cycle diseases¹¹. The elevated concentration of N-acetylaspartic acid in urine confirms a very rare case of CD. The LC-MS/MS diagnosis of CD was less tedious and less time-consuming than GC/MS method. The elevated concentration of homocysteine in plasma together with high levels of the amino acid, methionine, in DBS confirms cases of  homocystinuria. The elevated concentration of plasma L-pipecolic acid confirms patient with Zellweger Syndrome as a type of  peroxisomal disorders.

 

CONCLUSIONS:

The developed labeled internal standards LC-MS/MS method is a powerful technique for the diagnosis and confirmation of IMDs such as ethylmalonic aciduria, urea cycle ornithine carbomyltransferase deficiency, Canavan disease, homocystinuria and peroxisomal disorders. The method provides a simple, reliable and specific quantitation method for the diagnostic biomarkers of the above IMDs in different biological samples using small volumes and simple preparation procedures. The method is superior to GC/MS which involves time-consuming sample preparations, derivatization reactions and lengthy chromatographic analysis. The developed LC-MS/MS has a high degree of flexibility and can be tailored to satisfy physician’s requests.

 

ACKNOWLEGEMENT:

The authors highly appreciate the excellent collaboration of pediatricians and metabolic clinicians at the Government maternity and pediatrics hospitals, Kuwait for providing infants’ samples.

 

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Received on 12.04.2012       Modified on 01.05.2012

Accepted on 20.05.2012      © RJPT All right reserved