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Clinical Chemistry 54: 1451-1462, 2008. First published August 4, 2008; 10.1373/clinchem.2007.099226
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(Clinical Chemistry. 2008;54:1451-1462.)
© 2008 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Quantification of Carnitine and Acylcarnitines in Biological Matrices by HPLC Electrospray Ionization– Mass Spectrometry

Paul E. Minkler1, Maria S. K. Stoll1, Stephen T. Ingalls1, Shuming Yang1, Janos Kerner3 and Charles L. Hoppel1,2,a

Departments of1 Pharmacology, 2 Medicine, and 3 Nutrition, Case Western Reserve University School of Medicine, Cleveland, OH.

aAddress correspondence to this author at: Case Western Reserve University School of Medicine, Department of Pharmacology (4965), 10900 Euclid Ave., Cleveland, OH 44106. Fax (216) 368-5162; e-mail charles.hoppel{at}case.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: Analysis of carnitine and acylcarnitines by tandem mass spectrometry (MS/MS) has limitations. First, preparation of butyl esters partially hydrolyzes acylcarnitines. Second, isobaric nonacylcarnitine compounds yield false-positive results in acylcarnitine tests. Third, acylcarnitine constitutional isomers cannot be distinguished.

Methods: Carnitine and acylcarnitines were isolated by ion-exchange solid-phase extraction, derivatized with pentafluorophenacyl trifluoromethanesulfonate, separated by HPLC, and detected with an ion trap mass spectrometer. Carnitine was quantified with d3-carnitine as the internal standard. Acylcarnitines were quantified with 42 synthesized calibrators. The internal standards used were d6-acetyl-, d3-propionyl-, undecanoyl-, undecanedioyl-, and heptadecanoylcarnitine.

Results: Example recoveries [mean (SD)] were 69.4% (3.9%) for total carnitine, 83.1% (5.9%) for free carnitine, 102.2% (9.8%) for acetylcarnitine, and 107.2% (8.9%) for palmitoylcarnitine. Example imprecision results [mean (SD)] within runs (n = 6) and between runs (n = 18) were, respectively: total carnitine, 58.0 (0.9) and 57.4 (1.7) µmol/L; free carnitine, 44.6 (1.5) and 44.3 (1.2) µmol/L; acetylcarnitine, 7.74 (0.51) and 7.85 (0.69) µmol/L; and palmitoylcarnitine, 0.12 (0.01) and 0.11 (0.02) µmol/L. Standard-addition slopes and linear regression coefficients were 1.00 and 0.9998, respectively, for total carnitine added to plasma, 0.99 and 0.9997 for free carnitine added to plasma, 1.04 and 0.9972 for octanoylcarnitine added to skeletal muscle, and 1.05 and 0.9913 for palmitoylcarnitine added to skeletal muscle. Reference intervals for plasma, urine, and skeletal muscle are provided.

Conclusions: This method for analysis of carnitine and acylcarnitines overcomes the observed limitations of MS/MS methods.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Today, the usual method for analyzing carnitine and acylcarnitines uses butyl ester formation and tandem mass spectrometry (MS/MS) (1)(2)(3)(4). Because of its simplicity and rapidity, this method has been applied with great success to high-throughput newborn-screening programs (5)(6)(7)(8); however, limitations to this method have been identified. First, the preparation of acylcarnitine butyl esters partially hydrolyzes acylcarnitines. This hydrolysis produces artificially increased free carnitine values (9), and the basic procedure has been modified in response to this problem (10)(11)(12). Second, MS/MS is not sufficiently selective in some cases. False-positive test results due to the presence of isobaric contaminants have been reported (13)(14). Third, acylcarnitine constitutional isomers cannot be distinguished. This lack of specificity can lead to false-positive results due to contributions from other acylcarnitine species (15)(16).

We have developed HPLC methods for quantifying carnitine and acylcarnitines (17)(18)(19)(20). These assays possess several unique features: (a) sample isolation via weak cation exchange with silica gel; (b) derivatization with highly reactive reagents under mild reaction conditions (21)(22) that do not hydrolyze acylcarnitines, (c) HPLC for selective elution, and (d) detection by ultraviolet or fluorescence spectrophotometry. We reasoned that linking electrospray ionization–mass spectrometry (ESI-MS)1 detection to our HPLC method would permit highly specific detection of carnitine and acylcarnitines in biological specimens, and we have presented a detailed report on the conceptual basis for this strategy and the empirical characterization of its components (23). We report on the application of that procedure to the quantification of carnitine and acylcarnitines in biological samples.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
chemicals
Compounds labeled with stable isotopes were purchased from Cambridge Isotope Laboratories. More than 40 acylcarnitines were synthesized in our laboratory (24)(25)(26). We used Bovine Albumin Fraction V Solution (75 g/L in PBS; Invitrogen) as a proxy for biological samples. Solid-phase extraction columns (50-mg silica Bond Elut) were purchased from Varian. Other chemicals were obtained and used as previously described (23).

equipment
The HPLC-ESI-MS system consisted of an HP 1100 Series HPLC instrument (quaternary pump and degasser, column compartment, and autosampler) from Agilent Technologies and an LCQ Deca mass spectrometer from Thermo Finnigan. The HPLC column was 100 mm x 4.6 mm (inner diameter) and contained Hypersil MOS-1 C8, 3 µm (Thermo Hypersil-Keystone). Four chromatographic eluents were used (relative volumes): eluent A, acetonitrile/water (80/20); eluent B, acetonitrile/water (20/80); eluent C, acetonitrile/ water/acetic acid/triethylamine (20/80/0.5/0.5); and eluent D, acetonitrile/water/acetic acid/triethylamine (90/10/0.5/0.5) (23).

biological samples
The procedures followed were reviewed by the University Hospitals Case Medical Center Institutional Review Board. Human skeletal muscle, plasma, urine, blood, and blood spot samples were obtained from the Center for Inherited Disorders of Energy Metabolism (CIDEM, Cleveland, OH). Tissues (10–40 mg) were placed in 1 mL of ice-cold deionized water and homogenized for 30 s with an Omni 2000 tissue homogenizer (Omni International). Plasma was collected into tubes containing EDTA. Whole blood was collected by finger lance. Blood spots were 3/16-in disks punched from blood spotted to Schleicher & Schuell Grade 903 filter paper and dried; each disk was assumed to contain 7.6 µL of blood (4). Urine samples were routinely diluted with 4 volumes of water.

carnitine and acylcarnitine sample analysis
Carnitine solutions were calibrated (27), and stock solutions were prepared. Acylcarnitine solutions were calibrated with the calibrated carnitine solutions. The phosphate-buffered BSA solution (which is free of carnitine and acylcarnitines) replaced the biological sample in calibrators and QC samples. Calibrators and QC samples were then analyzed identically to biological samples.

analysis batches
We prepared samples in batches that contained different sample types (urine, plasma, skeletal muscle, and so forth), and each batch was completed in 3 days. Our average batch size was 24 samples, with the largest batch having 48 samples.

total and free carnitine
We added 50 µL of sample (tissue homogenate, plasma, urine, or other specimen) or 2 blood-spot disks to a microcentrifuge tube. We then added 50 µL of internal-standard solution (25 µmol/L of d3-carnitine) and 1 mL of acetonitrile/methanol solution (3 volumes/ 1 volume). Samples were vortex-mixed for 2 s (blood spots were placed on an orbital shaker for 30 min) and centrifuged (5 min at 13 200g). For measurement of free carnitine, we applied a 400-µL aliquot to a solid-phase extraction column. For total carnitine, we combined a 400-µL aliquot with 100 µL of 1 mol/L of KOH in methanol and placed the solution in a water bath at 50 °C for 60 min. After acylcarnitine hydrolysis, we transferred the sample to a microcentrifuge tube, added 50 µL of 1.5 mol/L phosphoric acid and 750 µL of acetonitrile/methanol solution (relative volumes, 3/1), centrifuged the tube for 5 min at 13 200g, and decanted the supernatant into a solid-phase extraction column.

acylcarnitines
We added 10 µL of sample (tissue homogenate, plasma, urine, or other specimen) or 1 blood-spot disk to a microcentrifuge tube. We then added 10 µL of internal-standard solution (50 µmol/L of d6-acetyl-, 5.0 µmol/L of d3-propionyl-, and 2.5 µmol/L each of undecanoyl-, undecanedioyl-, and heptadecanoylcarnitine) and 1 mL of acetonitrile/methanol (relative volumes, 3/1). We vortex-mixed the samples for 2 s (blood spots were placed on an orbital shaker for 30 min), centrifuged the samples, and decanted the supernatants into solid-phase extraction columns.

solid-phase extraction
Silica gel solid-phase extraction columns were prepared by washing with 0.5 mL of methanol (gravity flow was used throughout). Samples were applied to the columns, and the columns were washed with 0.5 mL methanol. We eluted carnitine and acylcarnitines with 1.0 mL of water/methanol/acetic acid solution (relative volumes, 10/9/1) and collected the effluents. We evaporated the effluents to dryness, reconstituted the samples in 200 µL of the 3/1 acetonitrile/ methanol solution described above, transferred them to HPLC autosampler vials, and then evaporated the vials to dryness.

derivatization
Each sample received N,N-diisopropylethylamine solution (10 µL in 10 mL acetonitrile) followed by pentafluorophenacyl trifluoromethanesulfonate solution (0.1 mol/L in acetonitrile). Samples received 50 µL of each reagent for total and free carnitine assays and 10 µL each for acylcarnitine assays. Samples were injected directly into the HPLC column (2 µL for total and free carnitine measurements and 5 µL for acylcarnitine measurements).

hplc
Acylcarnitine pentafluorophenacyl esters were separated by sequential ion-exchange/reversed-phase chromatography on a single non–end-capped C8 column (23). Elution began with 100% eluent A at a flow rate of 1.75 mL/min. At 2 min after injection, eluent A was replaced with 100% eluent B. At 4 min after injection, we instituted a gradient from 100% eluent C to 80% eluent C and 20% eluent D over a 3-min period. We then reduced the eluent flow rate to 0.50 mL/min and started a gradient to 70% eluent C and 30% eluent D over a 15-min period. We followed this step with a gradient to 50% eluent C and 50% eluent D over a 20-min period and then finished with a final gradient to 100% eluent D over 20 min. The system then maintained 100% eluent D for 8 min. At 70 min after injection, we increased the eluent flow rate to 1.75 mL/min and returned the system to 100% eluent A for an additional 5 min (total run time, 75 min per injection). We used an abbreviated version of this eluent system for analysis of total and free carnitine (total run time, 13.5 min per injection). Flow was diverted from the ion source to waste during the washing and equilibration phases (i.e., when the flow rate was 1.75 mL/min).

esi-ms
Carnitine and d3-carnitine pentafluorophenacyl esters (370.1 m/z and 373.1 m/z, respectively) were detected with 2 alternating selected reaction-monitoring scans and fragmented by the application of a relative collision energy of 30%. For acylcarnitines, we observed that the product ion spectra of the acylcarnitine pentafluorophenacyl esters have analogous patterns: generation of a compound-specific ion (molecular ion –59 m/z)+ and a common ion at 293 m/z. We performed full-scan mass spectrometry (with 30% source collision energy) and full-scan MS/MS sampling routines. We used full-scan mass spectrometry for qualitative and semiquantitative analyses and full-scan MS/MS for quantification.

quantification
For total and free carnitine, chromatographic peaks were integrated from the total ion current response and fit to linear equations. For acylcarnitines, we summed product ions [i.e., (molecular ion –59 m/z)+ and 293 m/z] to produce reconstructed ion chromatogram (RIC) peak signals, which were fit to quadratic equations.

detection and semiquantification of additional acylcarnitines
The collection of full-scan mass spectrometry spectra in acylcarnitine analysis qualitatively reveals unusual acylcarnitines and permits their semiquantification. For each biological sample, a qualitative acylcarnitine chromatogram was produced from the full-scan mass spectrometry data. We measured newly observed acylcarnitines by evaluating the RICs of their molecular ions (compared with the peak areas of one of the internal standards) and calculated their concentrations semiquantitatively by assuming that the response factors for the compound and the internal standard were identical.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The basic goal of this procedure is to augment the high-throughput screening analysis of acylcarnitines by addressing (a) the unintended hydrolysis of acylcarnitines, (b) false positives, and (c) isomer detection. In addition, quantification is carried out with multiple-point calibration curves. First, derivatization under mild conditions with pentafluorophenacyl trifluoromethanesulfonate does not hydrolyze acylcarnitines. Second, false positives can be reduced greatly by increasing the selectivity. We accomplished this objective with more selective sample-isolation and HPLC- separation procedures. The described cation-exchange solid-phase extraction process isolates both carnitine and acylcarnitines with high recoveries yet excludes potential interferences [e.g., this procedure removes palmitic acid (23)]. Other investigators who also have used cation-exchange solid-phase extraction have reported high recoveries of acylcarnitines from urine and plasma (28). The use of HPLC separation in acylcarnitine analysis before detection has been shown to distinguish false positives from true positives (29), and our sequential ion-exchange/reversed-phase HPLC method adds even more selectivity (23). We synthesized more than 40 acylcarnitines [e.g., cis-3,4-methylene- heptanoylcarnitine (26)] to be used as calibrators or internal standards and generated 42 individual multiple- point calibration curves (Figs. 1 and 2 ) for MS/MS quantification of these acylcarnitines. Fig. 1A displays both a representative free carnitine chromatogram for the 5.0-µmol/L point on the calibration curve and a calibration curve. The injection volume contained 10 pmol carnitine pentafluorophenacyl ester and 50 pmol d3-carnitine pentafluorophenacyl ester. The mass spectrometer monitored the transitions 370.1 m/z->311.0 m/z and 373.1 m/z->311.0 m/z, respectively. Fig. 1B displays a representative palmitoylcarnitine chromatogram for the 0.10-µmol/L point on the calibration curve. The injection volume contained 500 fmol palmitoylcarnitine pentafluorophenacyl ester and 25 pmol heptadecanoylcarnitine pentafluorophenacyl ester. The mass spectrometer monitored the transitions 608.3 m/z->product ion spectrum 165–650 m/z and 622.3 m/z->product ion spectrum 170–650 m/z, respectively. Also shown in Fig. 1B are the MS/MS full- scan spectra for palmitoylcarnitine and heptadecanoylcarnitine pentafluorophenacyl esters, along with the calibration curve that incorporated this point. Fig. 2 shows a representative acylcarnitine high QC injection (75.0 µmol/L acetylcarnitine, 15.0 µmol/L propionylcarnitine, and 1.50 µmol/L of each of the other acylcarnitines) including internal standards (50 µmol/L d6-acetylcarnitine, 5.0 µmol/L d3-propionylcarnitine, and 2.5 µmol/L each of undecanoyl-, undecanedioyl-, and heptadecanoylcarnitine). The chemical structures beneath the chromatogram illustrate a fragmentation of acylcarnitine pentafluorophenacyl esters consistent with the observed spectra. All of the acylcarnitine pentafluorophenacyl esters examined have similar product ion spectra that generate ions (molecular ion –59 m/z)+ and 293 m/z. The ion at 293 m/z is common to all acylcarnitine pentafluorophenacyl esters.


Figure 1
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  		Figure 1. Calibration curve chromatograms.

Free carnitine (A) and palmitoylcarnitine (B). The arrow in the calibration curves indicates the concentration corresponding to the chromatograms. A/D, analog-to-digital.


Figure 2
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  		Figure 2. Acylcarnitine QC chromatograms.

(A), Acylcarnitine pentafluorophenacyl ester common ion (293 m/z) RIC, generated from full-scan mass spectrometric sampling. (B), Superimposed RICs [(molecular ion –59 m/z)+ and 293 m/z] generated from full-scan MS/MS scans for the different acylcarnitine pentafluorophenacyl esters. (C), Separated RICs generated from full-scan MS/MS scans [(molecular ion –59 m/z)+ and 293 m/z] for the different acylcarnitine pentafluorophenacyl esters. Peak identities are as follows: 1, acetyl-; 2, d6-acetyl-; 3, 3-hydroxy-isovaleryl-; 4, propionyl-; 5, d3-propionyl-; 6, isobutyryl-; 7, butyryl-; 8, tigloyl-; 9, 3-methyl-crotonyl-; 10, 2-methyl-butyryl-; 11, isovaleryl-; 12, valeryl-; 13, benzoyl-; 14, phenylacetyl-; 15, hexanoyl-; 16, phenylpropionyl-; 17, succinyl-; 18, methylmalonyl-; 19, 4-methyl-hexanoyl-; 20, 4-phenyl-butyryl-; 21, cis-3,4-methylene-heptanoyl- [Yang et al. (26)]; 22, glutaroyl-; 23, ethylmalonyl-; 24, valproyl-; 25, octanoyl-; 26, adipoyl-; 27, 3-methyl-glutaroyl-; 28, 5-decynoyl-; 29, suberoyl-; 30, cis-3,4-methylene-nonanoyl-; 31, cis-4-decenoyl-; 32, sebacoyl-; 33, 4-methyl-octanoyl-; 34, decanoyl-; 35, trans-2-dodecenoyl-; 36, undecanoyl-; 37, undecanedioyl-; 38, trans-2-tetradecenoyl-; 39, lauroyl-; 40, myristoyl-; 41, palmitoleoyl-; 42, trans-2-hexadecenoyl-; 43, linoleoyl-; 44, palmitoyl-; 45, oleoyl-; 46, heptadecanoyl-; 47, stearoylcarnitine pentafluorophenacyl ester.

validation studies
Extraction efficiency.
We assessed recoveries from the sample-isolation procedure by comparing results obtained with calibrators carried through the entire procedure with results obtained for identical calibrators that were simply evaporated. We added internal- standard solutions just before derivatization, evaluated chromatographic peak area ratios for compounds and internal standards, and calculated recoveries. Example results [mean (SD), n = 6] are as follows: total carnitine, 69.4% (3.9%); free carnitine, 83.1% (5.9%); acetylcarnitine, 102.2% (9.8%); palmitoylcarnitine, 107.2% (8.9%). The recovery of dicarboxylic acylcarnitines was not as high as for the other acylcarnitines [e.g., glutaroylcarnitine, 58.6% (7.7%)] because of the second carboxylate functional group on these molecules. To accommodate this lower recovery, we use a dicarboxylic acylcarnitine (undecanedioylcarnitine) as the internal standard for these compounds.

Standard-addition studies.
Fig. 3 summarizes the results of standard-addition studies with carnitine and representative acylcarnitines. Increasing amounts of carnitine or an acylcarnitine added to biological samples display a linear relationship with the detected carnitine or acylcarnitine, with all the slopes and linear regression coefficient values very close to 1. Fig. 3B shows that adding increasing amounts of acetylcarnitine to a plasma sample does not change the free carnitine concentration. On the other hand, the amount of acetylcarnitine detected is directly proportional to the added acetylcarnitine. This result proves that acetylcarnitine is not hydrolyzed to carnitine with this method. We added increasing concentrations of the 42 acylcarnitines to aliquots of samples of nonpathologic human plasma, urine, and homogenized skeletal muscle and assayed for acylcarnitines. The results of the analyses for octanoylcarnitine and palmitoylcarnitine are shown in Fig. 3 , C and D.


Figure 3
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  		Figure 3. Standard-addition study.

(A), We added increasing concentrations of carnitine to aliquots of a nonpathologic human plasma and analyzed the aliquots for total and free carnitine. (B), Increasing concentrations of acetylcarnitine were added to aliquots of nonpathologic human plasma, and the aliquots were analyzed for total and free carnitine and acetylcarnitine. Increasing concentrations of the 42 acylcarnitines were added to aliquots of nonpathologic human plasma, urine, and homogenized skeletal muscle samples, and the samples were analyzed for acylcarnitines. (C), Added octanoylcarnitine. (D), Added palmitoylcarnitine.

Imprecision assessment with biological samples.
We assessed the imprecision of the method via multiple analyses of identical aliquots of pooled samples of nonpathologic human plasma. Example within-batch results (n = 6) and between-batch results (n = 18) [mean (SD)] are respectively as follows: total carnitine, 58.0 (0.9) and 57.4 (1.7) µmol/L; free carnitine, 44.6 (1.5) and 44.3 (1.2) µmol/L; acetylcarnitine, 7.74 (0.51) and 7.85 (0.69) µmol/L; and palmitoylcarnitine, 0.12 (0.01) and 0.11 (0.02) µmol/L.

QC tolerance limits.
We analyzed 3 QC samples (low, medium, and high concentrations) as a part of each analysis batch. The values were 5.00, 50.0, and 400 µmol/L for free carnitine and total carnitine; 10.0, 35.0, and 75.0 µmol/L for acetylcarnitine; and 1.00, 7.00, and 15.0 µmol/L for propionylcarnitine. For all other acylcarnitines, the values were 0.20, 0.70, and 1.50 µmol/L. We evaluated the percent differences from expected values. The tolerance limits for free carnitine, total carnitine, and each of the 42 acylcarnitines for the low QC concentration were within ±20% of expected values. The tolerance limits for free carnitine, total carnitine, and each of the 42 acylcarnitines for the medium and high QC concentrations were within ±15% of expected values. Typically, within-batch analyses met >90% of the 132 tolerance-limits tests.

Calibration curve tolerance limits.
All points on the calibration curves for total and free carnitine (13 calibration points for each curve) and the 42 acylcarnitine calibrators (7 calibration points for each compound, excluding the zero point) were back-calculated, and these values were compared with expected values. The tolerance limit for the percent difference was within ±20% of expected values for the lowest concentration and within ±15% of expected values for all the other concentrations. Typically, within-batch analyses met >90% of the 320 tolerance-limits tests.

application to patient samples
"Balance" studies.
We calculated the total acylcarnitine concentration (Sum) by adding the concentration values for the individual acylcarnitines and calculated the difference between total and free carnitine concentrations (Total-free). The Sum/Total-free ratio will have a value of 1 if all acylcarnitines have been accounted for. Table 1 presents the concentrations and results of "balance" studies obtained with this procedure for representative patient samples. The patients with methylmalonic aciduria, glutaric acidemia type I, and isovaleric acidemia were receiving carnitine as a part of their treatment. The whole-blood sample and the blood spot were from a single healthy volunteer and were collected at the same time. The close agreement in concentrations demonstrates the precision of this procedure, despite the error expected for the sampling procedure of collecting blood onto filter paper (30).


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  		Table 1. Measured concentrations for selected samples, including the results of balance studies.

Fig. 4A shows the separation and detection of valproylcarnitine and octanoylcarnitine in urine from a patient receiving valproate therapy. The top chromatogram is the mass spectrometry qualitative chromatogram. The middle chromatogram is the MS/MS quantitative chromatogram for valproyl- and octanoylcarnitine in the patient’s urine. The bottom chromatogram is a calibrator analyzed during the same batch. These RICs were constructed from full MS/MS scans for C8 acylcarnitine pentafluorophenacyl esters (496.2 m/z->product ion spectrum 135–550 m/z). The inset spectra are the product ion mass spectra for valproyl- and octanoylcarnitine pentafluorophenacyl esters. We observed that the product ion mass spectra for valproyl- and octanoylcarnitine pentafluorophenacyl esters have identical features and are essentially indistinguishable. Therefore, although we used MS/MS to detect these acylcarnitines, the identification of these acylcarnitines rests with the chromatographic system rather than the mass spectrometer. The results of the balance study for this urine sample are shown in Table 1Up . Our examination of the mass spectrometry common ion chromatogram revealed several chromatographic peaks that are not among those for which we have reference compounds. These peaks include those for 2 isomeric forms of C9 acylcarnitine and 2 isomeric forms of C9:1 acylcarnitine. The semiquantified concentrations obtained for these compounds are as follows: C9a, 2.36 µmol/g creatinine; C9b, 0.55 µmol/g; C9:1a, 0.61 µmol/g; and C9:1b, 1.93 µmol/g. Inclusion of these values in the calculation increases the Sum/Total-free ratio from 0.40 to 0.45; however, the difference between the implicit acylcarnitine content (Total-free) and what can be accounted for (Sum) is still substantial. This difference does not occur in all urine samples (e.g., the Sum/Total-free ratio for the patient with isovaleric acidemia is 0.99; Table 1Up ), but many urine samples contain acylcarnitines in addition to those for which we can account.


Figure 4
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  		Figure 4. Chromatograms of patient samples.

(A), Patient receiving valproate therapy. The top chromatogram is the mass spectrometry (MS) qualitative chromatogram. Peak labels are the same as in Fig. 2Up . The inset image is the mass spectrum for valproylcarnitine pentafluorophenacyl ester (496.2 m/z). The middle chromatogram is the MS/MS quantitative chromatogram for valproyl- and octanoylcarnitine pentafluorophenacyl esters. The bottom chromatogram is from a calibration curve point analyzed during the same batch. The inset spectra are the product ion mass spectra for valproyl- and octanoylcarnitine pentafluorophenacyl esters. (B), Separation and detection of isovalerylcarnitine in urine from a patient with isovaleric acidemia. The top chromatogram is the MS qualitative chromatogram. The middle chromatogram is the MS/MS quantitative chromatogram for isovalerylcarnitine pentafluorophenacyl ester. The bottom chromatogram is from a calibration curve point analyzed during the same batch.

Fig. 4BUp shows the separation and detection of isovalerylcarnitine in urine from a patient with isovaleric acidemia. The top chromatogram is the mass spectrometry qualitative chromatogram. The middle chromatogram is the MS/MS quantitative chromatogram for isovalerylcarnitine pentafluorophenacyl ester. The bottom chromatogram is from a calibrator analyzed during the same batch, showing 2-methyl-butyrylcarnitine, isovalerylcarnitine, and valerylcarnitine pentafluorophenacyl esters. The product ion mass spectra are essentially indistinguishable. Therefore, although the detection of these acylcarnitines is by MS/MS, the identification of these acylcarnitines rests with the chromatographic system rather than the mass spectrometer. Table 1Up presents the quantitative results and the results of the balance study for this sample.

Fig. 5 shows the separation and detection of long-chain acylcarnitines in plasma from a patient with deficiency of long-chain 3-hydroxy-acyl-CoA dehydrogenase. In Fig. 5A , the top chromatogram is the mass spectrometry qualitative chromatogram, and the remaining chromatograms are the MS/MS quantitative chromatograms. These RICs [(molecular ion –59 m/z)+ and 293 m/z] were generated from full-scan MS/MS spectra of the molecular ions for trans-2-tetradecenoyl-, myristoyl-, palmitoleoyl-, trans-2-hexadecenoyl-, linoleoyl-, palmitoyl-, oleoyl-, heptadecanoyl-, and stearoylcarnitine pentafluorophenacyl esters. We observed isomeric forms of C14:1 acylcarnitines in addition to trans-2-tetradecenoylcarnitine and observed another C18:1 acylcarnitine in addition to oleoylcarnitine. Fig. 5B shows the mass spectrometry semiquantitative chromatograms, which reveal 2 isomeric forms each of hydroxy-C16:0, hydroxy-C18:1, and hydroxy-C18:0 acylcarnitines that are present in this plasma sample. Measured values for this sample were as follows: total carnitine, 77.8 µmol/L; free carnitine, 52.9 µmol/L; acetylcarnitine, 8.83 µmol/L; propionylcarnitine, 0.48 µmol/L; hexanoylcarnitine, 0.13 µmol/L; octanoylcarnitine, 0.13 µmol/L; decanoylcarnitine, 0.24 µmol/L; lauroylcarnitine, 0.81 µmol/L; trans-2-dodecenoylcarnitine, 0.15 µmol/L; myristoylcarnitine, 0.30 µmol/L; trans-2-tetradecenoylcarnitine, 0.18 µmol/L; palmitoleoylcarnitine, 0.43 µmol/L; palmitoylcarnitine, 0.31 µmol/L; oleoylcarnitine, 0.44 µmol/L; stearoylcarnitine, 0.11 µmol/L; and other acylcarnitines, 1.28 µmol/L. We quantified the additional isomeric forms of C14:1 acylcarnitine with the trans-2-tetradecenoylcarnitine calibration curve (C14:1a, 0.53 µmol/L; C14:1b, 0.14 µmol/L) and quantified the other isomeric form of C18:1 acylcarnitine with the oleoylcarnitine calibration curve (C18:1a, 0.33 µmol/L). We also obtained semiquantitative values for the other observed acylcarnitines: hydroxy-C16:0a, 0.40 µmol/L; hydroxy-C16:0b, 0.18 µmol/L; hydroxy-C18:1a, 0.72 µmol/L; hydroxy-C18:1b, 0.59 µmol/L; hydroxy-C18:0a, 0.53 µmol/L; hydroxy-C18:0b, 0.23 µmol/L. The results of the balance study were as follows: Total-free, 24.9 µmol/L; Sum, 17.5 µmol/L; Sum/Total-free ratio, 0.70. See the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol54/issue9 for the reference intervals for plasma, urine, and skeletal muscle generated with this procedure.


Figure 5
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  		Figure 5. Patient with deficiency in long-chain 3-hydroxy-acyl-CoA dehydrogenase.

(A), The top chromatogram is the mass spectrometry (MS) qualitative chromatogram, and the remaining chromatograms are the MS/MS quantitative chromatograms. (B), MS semiquantitative chromatograms.


   Acknowledgments
 
Grant/Finding Support: This research was supported in part by NIH Road Map Grant 1R33DK070291-01, NIH Program Project Grants AG15885 and HL 074237, and NIH DK066107.

Financial Disclosures: The samples described were analyzed through the Center for Inherited Disorders of Energy Metabolism (CIDEM), and the authors were financially compensated for this service.

Acknowledgments: We thank the Center for Inherited Disorders of Energy Metabolism (CIDEM, Cleveland, OH) for the human biopsy samples and patient urine and plasma samples. Portions of this manuscript appeared as Poster 45 at the Society for Inherited Metabolic Disorders 2007 Annual Meeting, Nashville, Tennessee, March 25–28, 2007.


   Footnotes
 
1 Nonstandard abbreviations: ESI-MS, electrospray ionization–mass spectrometry; MS/MS, tandem or recursive mass spectrometry; RIC, reconstructed ion chromatogram.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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