Analysis of Acylcarnitines in Dried Blood Spots (DBS) Samples by FIA-MS/MS

Arun Babu Kumar Ph.D.¹, Sarah Aijaz Ph.D.¹, Uma Sreenivasan Ph.D.¹ Merck ¹ Round Rock, TX, USA

Workflow

Extraction

Sample extraction and derivatization

Analysis

FIA-MS/MS Analysis

Detection

Detection & Quantification

1. Overview: FIA-MS/MS for Acylcarnitines Analysis in Dried Blood Spots (DBS)
2. Detailed Acylcarnitine FIA-MS/MS Workflow
   • 2.1 Acylcarnitine SIL Internal Standards
   • 2.2 Acylcarnitine Sample Extraction and Derivatization steps
   • 2.3 DBS Extraction Solution Preparation
   • 2.4 Extraction and Derivatization of DBS punch
   • 2.5 FIA-MS/MS conditions
   • 2.6 Acquisition Parameters for Derivatized Acylcarnitines
3. Acylcarnitine FIA-MS/MS Quantification and Results
   • 3.1 MRM Chromatogram of SIL Internal Standards
   • 3.2 MRM Chromatogram of Blank and Control Injections
   • 3.3 Quantification of Acylcarnitines and Interpretation of Results
4. Conclusion: Quantitative Analysis of Acylcarnitines from Dried Blood Spots

1. Overview: FIA-MS/MS for Acylcarnitines Analysis in Dried Blood Spots (DBS)

In the 1960s, Robert Guthrie implemented the large-scale use of dried blood spots (DBS) for the screening of newborns for Phenylketonuria (PKU). Since then, the use of DBS in diagnosis has expanded to include a variety of metabolic disorders and other ailments. Tandem mass spectrometry (MS/MS) has developed to be the choice of technique to analyze the DBS samples due to its sensitivity and capacity to multiplex to quantify several analytes simultaneously. MS/MS is widely used in clinical labs for the analysis of acylcarnitine panel in DBS to diagnose several fatty acid oxidation disorders and organic acid disorders.^1-3 Flow injection analysis-MS/MS (FIA-MS/MS) is widely used in Tier-1 testing for identification and quantitation of acylcarnitines by direct quantitation against suitable internal standards. ^4,5 Figure 1 below shows the overall workflow for the FIA-MS/MS analysis.

Figure 1. Workflow for acylcarnitine analysis in dried blood spots (DBS) by FIA-MS/MS.

Prepare Extraction Solution with SIL internal standard	🠦	Punch 3.2 mm sample of DBS	🠦	Extract DBS punch with Extraction solution	🠦	Supernatent dried under nitrogen stream	🠦	Residue derivatized to butyl esters	🠦	Dried under stream of nitrogen	🠦	Residue reconstituted to solution for FIA-MS/MS

Quantification of acylcarnitines using stable isotope-labeled (SIL) internal standards

This article illustrates the utility of accurate and convenient stable isotope-labeled (SIL) internal standard solutions from Supelco for the analysis of acylcarnitine panel by FIA-MS/MS. The solution-based SIL internal standards are certified reference materials (CRMs), eliminating the need for quantitative transfer of lyophilized internal standards prior to dilution and provide accuracy and flexibility to prepare only the required amount of extraction solution with SIL internal standards.

Acylcarnitine DBS reference samples from CDC were used as controls and two levels of control DBS samples from Recipe ClinChek with known concentrations for 13 acylcarnitines were used as samples for the analysis.

2. Detailed Acylcarnitine FIA-MS/MS Workflow

2.1 Acylcarnitine SIL Internal Standards

Stable isotope-labeled (SIL) Acylcarnitine CRM solutions with 13 different heavy atom labeled acylcarnitines were used for the accurate quantification of different acylcarnitine species with the most closely related internal standard. Stable isotope-labeled CRMs in solution form offer greater flexibility, accuracy, homogeneity, and convenience in use than lyophilized materials. CRMs solution eliminates the need for quantitative transfer of lyophilized content for dilution to DBS extraction solution. Figure 2 shows the chemical structures, isotope labeling, and concentrations of the CRMs used as internal standards for the acylcarnitines analysis.

Figure 2. Chemical structures of internal standards used for the acylcarnitines analysis.

2.2 Acylcarnitine Sample Extraction and Derivatization steps

Table below shows the sequential steps involved in the extraction of acylcarnitines from DBS samples and derivatization into their butyl esters for enhanced sensitivity in the MS/MS.

Extraction of DBS Punch

Using an 1/8-inch hole puncher a DBS spot is punched out into a 1.5 mL microcentrifuge tube	🠦	100 µL of the extraction solution with SIL internal standards is assed to the DBG punch	🠦	Spin the sample tubes on a centrifuge and 95 µL of the spernatant to a new tube	🠦	The solution is concenrated to dryness under a stream of nitogen

Derivatization of DBS extract

Figure 3. DBS sample extraction and derivatization steps

Add 100 µL of 3 M HCI in 1-butanol and vortex	🠦	Incubate at 60° C for 30 minutes	🠦	The contents of the tube is dried under a steam of nitrogen	🠦	Vortex the derivatized residue methanol/water (80:20) mixture	🠦	Centrifuge the solution and supernatant was analyzed by FIA-MS/MS

2.3 DBS Extraction Solution Preparation

The DBS extraction solution can be conveniently prepared by combining and diluting an appropriate amount of A-148-1ML, A-147-1ML, A-163 -1ML, A-181-1ML, and A-164-1ML in a volumetric flask with methanol (Figure 4). The concentrations of SIL acylcarnitines in the DBS extraction solution thus prepared are given in Table 1.

Step	Preparation of DBS Extraction Solution with SIL Acylcarnitine Internal Standards
1	Sonicate the SIL Acylcarnitines solution ampules at room temperature for 5 minutes and mix by vortexing
2	Using a positive displacement pipette transfer 32 µL of A-148-1ML, 30 µL of A-147-1ML, 10 µL of A-163 -1ML, 75 µL of A-181-1ML and 20 µL of A-164-1ML to a 250 mL Volumetric flask containing about 100 mL of LCMS grade methanol
3	Make up the volume of the 250 mL Volumetric flask with LCMS grade methanol and mix well. Store this extraction solution according to labeling guidance

Figure 4. Dilution schematic to prepare DBS extraction solution

Table 1. Concentration of SIL acylcarnitines in DBS extraction solution

SigmaAldrich Part. No	SIL Carnitine	Volume of CRM soln (µL) diluted to 250 mL	Conc. of CRM solution		Conc. of DBS Extraction Solution
			µg/mL	µM	µM	mg/L
A-148-1ML	C0-Carnitine-D9	32	1000	5873	0.7518	0.128
A-148-1ML	C2-Carnitine-D3	32	1000	4848	0.6206	0.128
A-163-1ML	C3-Carnitine-D3	10	501	2275	0.0910	0.020
A-147-1ML	C4-Carnitine-D3	30	100	426.8	0.0512	0.012
A-147-1ML	iC5-Carnitine-D9	30	100	393.1	0.0472	0.012
A-181-1ML	C5DC-Carnitine-D3	75	100	360.6	0.1082	0.030
A-147-1ML	C6-Carnitine-D3	30	100	381.2	0.0457	0.012
A-147-1ML	C8-Carnitine-D3	30	100	344.3	0.0413	0.012
A-147-1ML	C10-Carnitine-D3	30	100	314.0	0.0377	0.012
A-147-1ML	C12-Carnitine-D9	30	100	283.6	0.0340	0.012
A-147-1ML	C14-Carnitine-D9	30	100	262.7	0.0315	0.012
A-164-1ML	C16-Carnitine-D3	20	500	1242	0.0994	0.040
A-147-1ML	C18-Carnitine-D3	30	200	464.4	0.0557	0.024

2.4 Extraction and Derivatization of DBS punch

Extraction steps of DBS punch is given below.

Step	Extraction of DBS punch
1	DBS collection card without blood was used as a blank sample and 4 levels of DBS reference standards from CDC were used as control samples. Additionally, 2 levels of DBS standards from Recipe ClinChek-Control (Part# MS10182) were also extracted as reference samples. Blank and controls were extracted as detailed in the steps below.
2	Using a 1/8 inch single hole puncher a DBS spot is punched out into a 1.5 mL microcentrifuge tube.
3	100 µL of the Extraction solution with SIL internal standards is added to the DBS punch and shaken on a rocker for 30 minutes at room temperature.
4	Spin the sample tubes on a microcentrifuge and 95 µL of the supernatant is transferred to a new microcentrifuge tube and the solution is concentrated to dryness under a stream of nitrogen.

The table below describes the steps involved in the Derivatization of DBS extract for enhanced sensitivity in MS.

Step	Derivatization of DBS extract
1	Add 100 µL of 3M HCl in 1-butanol to the dried extract residue and vortex at 1200 rpm for 1 minute
2	Incubate at 60˚C for 30 minutes followed by spinning the sample tubes on a microcentrifuge
3	The contents of the tube are dried under a stream of nitrogen
4	Vortex the derivatized residue with 100 µL of methanol/water (80:20) mixture at 1500 rpm for 2 minutes
5	Spin the resulting solution on a microcentrifuge and 95 µL of the supernatant was transferred to an HPLC autosampler vial with vial inserts

2.5 FIA-MS/MS conditions

Experimental parameters for FIA-MS/MS:

LC-MS/MS
Column:	None
Detection:	MRM Acquisition with Electrospray Ionization in Positive Mode
Instrumentation:	Agilent 1290 Infinity II coupled to Ultivo TQ
Mobile phase A:	0.1% Formic acid in LC/MS Grade Water
Mobile phase B:	0.1% Formic acid in LC/MS Grade Acetonitrile
Injection Volume	1 µL
Analysis Time	3 minutes

HPLC Gradient conditions:

Step	Time (min)	Mobile Phase A%	Mobile Phase B%	Flow rate
1	0.00	50	50	0.05 mL/min
2	2.00	50	50	0.05 mL/min
3	3.00	50	50	0.15 mL/min

MS Source Parameters:

Source Conditions	Value (+)
Gas Temp (°C)	275
Gas Flow (L/min)	12.0
Nebulizer (psi)	45
Sheath Gas Temperature (°C)	300
Sheath Gas Flow (L/min)	10.0
Capillary Voltage	4000
Nozzle Voltage	0

2.6 Acquisition Parameters for Derivatized Acylcarnitines

Table 2. MS/MS MRM acquisition parameters for each analyte and internal standards

Compound	Analyte/ISTD	Precursor (m/z)	Product (m/z)	Dwell (ms)	Fragmentor (V)	CE (V)	Polarity
BuEster C0	Analyte	218.2	85	30	100	27	Positive
BuEster C0 d9	ISTD	227.2	85	30	100	27	Positive
BuEster C2	Analyte	260.2	85	30	100	22	Positive
BuEster C2 d3	ISTD	263.2	85	30	100	22	Positive
BuEster C3:0	Analyte	274.2	85	30	105	22	Positive
BuEster C3:0 d3	ISTD	277.2	85	30	105	22	Positive
BuEster C4:0	Analyte	288.2	85	30	115	24	Positive
BuEster C4:0 d3	ISTD	291.2	85	30	115	24	Positive
BuEster C5:1	Analyte	300.3	85	30	115	26	Positive
BuEster C5:0	Analyte	302.2	85	30	115	26	Positive
BuEster C4OH	Analyte	304.2	85	30	115	24	Positive
BuEster iC5:0 d9	ISTD	311.3	85	30	115	26	Positive
BuEster C6:0	Analyte	316.2	85	30	115	26	Positive
BuEster C5OH	Analyte	318.3	85	30	115	26	Positive
BuEster C6:0 d3	ISTD	319.2	85	30	115	26	Positive
BuEster C8:0	Analyte	344.3	85	30	125	28	Positive
BuEster C8:0 d3	ISTD	347.3	85	30	125	28	Positive
BuEster C3DC	Analyte	360.3	85	30	125	30	Positive
BuEster C10:1	Analyte	370.3	85	30	135	28	Positive
BuEster C10:0	Analyte	372.3	85	30	135	28	Positive
BuEster C10:0 d3	ISTD	375.3	85	30	135	28	Positive
BuEster C5DC	Analyte	388.2	85	30	125	30	Positive
BuEster C5DC d3	ISTD	391.3	85	30	125	30	Positive
BuEster C12:1	Analyte	398.3	85	30	135	30	Positive
BuEster C12:0	Analyte	400.3	85	30	135	30	Positive
BuEster C12:0 d9	ISTD	409.4	85	30	135	30	Positive
BuEster C14:2	Analyte	424.3	85	30	135	32	Positive
BuEster C14:1	Analyte	426.3	85	30	135	32	Positive
BuEster C14:0	Analyte	428.4	85	30	135	32	Positive
BuEster C14:0 d9	ISTD	437.4	85	30	135	32	Positive
BuEster C16:1	Analyte	454.4	85	30	135	34	Positive
BuEster C16:0	Analyte	456.4	85	30	135	34	Positive
BuEster C16:0 d3	ISTD	459.4	85	30	135	34	Positive
BuEster C16OH	Analyte	472.4	85	30	135	34	Positive
BuEster C18:2	Analyte	480.3	85	30	135	35	Positive
BuEster C18:1	Analyte	482.4	85	30	135	35	Positive
BuEster C18:0	Analyte	484.5	85	30	135	35	Positive
BuEster C18:0 d3	ISTD	487.5	85	30	135	35	Positive
BuEster C18OH	Analyte	500.5	85	30	135	35	Positive

3. Acylcarnitine FIA-MS/MS Quantification and Results

3.1 MRM Chromatogram of SIL Internal Standards

Figure 5. MRM trace for butyl ester derivatized SIL acylcarnitines C0 d9, C10:0 d3, C12:0 d9, C14:0 d9, C16:0 d3, C18:0 d3, C2 d3 and C3 d3

Figure 6. MRM trace for butyl ester derivatized SIL acylcarnitines C4 d3, C5DC d3, C6 d3, C8:0 d3 and iC5 d9

3.2 MRM Chromatogram of Blank and Control Injections

Overlap chromatograms for all MRM transitions that include SIL internal standards and native acylcarnitines are shown in Figure 5. In the first overlapped MRM chromatogram, the control blank injection has a baseline signal for injection of methanol-water mixture. Whereas the second MRM overlap shows the injection of DBS extract from a CDS control sample.

Blank (80:20 methanol/water) injections: Shows no background Interferences for all analytes and SIL Internal Standards

Figure 7. MRM traces for all analytes and SIL internal standards with a blank injection of 80:20 methanol/water (top) and CDC Level-D DBS control (bottom)

3.3 Quantification of Acylcarnitines and Interpretation of Results

Acylcarnitine concentration is determined by comparing the MRM signal intensities against the know concentration of closely related SIL internal standards using the formula in the table below.

Figure 8. Formula to calculate acylcarnitine concentration in DBS sample

CA = (AA x VEX x CIS) ÷ (VB x AIS)		CA Target analyte concentration in blood (µM) AA Area count for analyte VEX Volume of Extraction solution (µL) CIS Concentration of SIL internal standard in Extraction solution (µM) VB Volume of blood in DBS punch (µL) (3.1 µL for 3.2 mm DBS punch AIS Area count for SIL internal standard

Accuracy was demonstrated through quantification of the four levels of control DBS samples from CDC with the known concentration of 20 acylcarnitines. The samples were analyzed (minimum of 3 replicate DBS extractions for each level) and the measured values were plotted against the actual mean concentrations of the control standards.⁶ As shown in Table 3, the correlation (slope) and R² is close to 1 for all acylcarnitines, indicative of the good accuracy of the quantification. The correlations are shown graphically in Figures 9 and 10.

In addition, two levels of control DBS samples from Recipe ClinChek with known concentration for 13 acylcarnitines were analyzed (in 6 replicate extractions for each level). As indicated in Table 4, the measured concentrations of all the acylcarnitines were within the expected control range.

Evaluation of measured acylcarnitines concentration vs. actual concentration in CDC control DBS Samples

Table 3. Correlation (slope) between the measured concentration and the actual mean concentration of acylcarnitines in DBS control standards from CDC

Analyte	Slope	R2	Intercept
C0	1.1	0.9941	-3.175
C2	0.8	0.9930	-0.873
C3	0.8	0.9964	-0.128
C3DC	1.0	0.9949	-0.062
C4:0	0.8	0.9927	0.083
C4OH	0.8	0.9955	-0.015
C5:0	0.9	0.9977	-0.035
C5:1	1.0	0.9974	-0.054
C5DC	0.9	0.9968	-0.040
C5OH	0.8	0.9961	-0.013
C6:0	0.8	0.9987	-0.009
C8:0	0.8	0.9977	-0.034
C10:0	0.7	0.9982	-0.028
C12:0	0.8	0.9986	-0.036
C14:0	0.8	0.9993	-0.025
C14:1	0.9	0.9988	-0.028
C16:0	0.9	0.9982	-0.108
C16OH	0.8	0.9992	-0.004
C18:0	0.9	0.9984	-0.056
C18OH	0.9	0.9986	-0.008

Evaluation of measured acylcarnitines concentration vs. actual concentration in Recipe control DBS samples

Table 4. Comparison of the average measured concentration of acylcarnitines against the expected concentration range in the Recipe ClinChek control DBS samples

Acylcarnitine	Control Level	Average Measured Concentration (µM)	Expected Control Range (µM)
C0	Level-I	26.1	11.7 - 40.4
	Level-II	170	65.9 - 198
C2	Level-I	11.1	3.37 - 15.9
	Level-II	76.2	29.9 - 119
C3	Level-I	1.86	1.59 - 3.30
	Level-II	12.7	10.2 - 19.0
C4	Level-I	0.753	0.645 - 1.34
	Level-II	6.92	5.90 - 11.0
C5	Level-I	0.478	0.271 - 0.713
	Level-II	2.34	1.31 - 3.06
C5DC	Level-I	0.552	0.462 - 1.85
	Level-II	2.46	1.98 - 6.82
C6	Level-I	0.366	0.260 - 0.541
	Level-II	1.08	0.748 - 1.39
C8:0	Level-I	0.374	0.225 - 0.675
	Level-II	2.31	1.39 - 3.67
C10:0	Level-I	0.196	0.112 - 0.336
	Level-II	1.04	0.593 - 1.56
C12:0	Level-I	0.406	0.262 - 0.544
	Level-II	6.61	3.89 - 7.23
C14:0	Level-I	0.421	0.270 - 0.711
	Level-II	3.42	2.12 - 4.94
C16:0	Level-I	0.986	0.661 - 1.98
	Level-II	9.22	5.82 - 15.3
C18:0	Level-I	0.524	0.327 - 0.982
	Level-II	4.04	2.37 - 6.26

Correlation plots between measured and actual acylcarnitines concentration in CDC control DBS samples

Figure 9. Correlation plot for the comparison of measured concentrations of acylcarnitines (C0, C2, C3, C3DC, C4, C4OH, C5:0, C5:1, C5DC) against the expected concentrations (MS/MS non-kit) in the four levels of CDC DBS controls

Figure 10. Correlation plot for the comparison of measured concentrations of acylcarnitines (C5OH, C6:0, C8:0, C10:0, C12:0, C14:0, C14:1, C16:0, C16OH, C18:0, C18OH) against the expected concentrations (MS/MS non-kit) in the four levels of CDC DBS controls

4. Conclusion: Quantitative Analysis of Acylcarnitines from Dried Blood Spots

This study illustrates the use of stable isotope-labeled (SIL) Acylcarnitines Certified Reference Materials (CRM) for the quantitation of acylcarnitines in dried blood spots. FIA-MS/MS analysis in combination with the SIL Acylcarnitine CRM solutions from Supelco can be used effectively to accurately measure the concentration of different acylcarnitine species in dried blood spots (DBS). Due to the availability of a wide diversity of SIL acylcarnitine species from Supelco, the SIL Acylcarnitine CRM that is most chemically similar to the analyte could be chosen as the MS/MS reference for the utmost accurate quantification. The SIL CRMs in solutions offer greater flexibility, accuracy, homogeneity, and convenience than lyophilized materials. Solutions allow flexibility to prepare any volume of DBS extraction solution with the reference materials, as desired by the analyst. The CRMs in solution form eliminate the need for quantitative transfer of the lyophilized content for dilution to DBS Extraction Solution. A complete series of native Acylcarnitines Mix CRM solutions are also available from Supelco for instrument calibration, controls, and MS/MS tuning.

References

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2. Zytkovicz TH, Fitzgerald EF, Marsden D, Larson CA, Shih VE, Johnson DM, Strauss AW, Comeau AM, Eaton RB, Grady GF. 2001. Tandem Mass Spectrometric Analysis for Amino, Organic, and Fatty Acid Disorders in Newborn Dried Blood Spots. 47(11):1945-1955. https://doi.org/10.1093/clinchem/47.11.1945

3. Chace DH, Lim T, Hansen CR, De Jesus VR, Hannon WH. 2009. Improved MS/MS analysis of succinylacetone extracted from dried blood spots when combined with amino acids and acylcarnitine butyl esters. Clinica Chimica Acta. 407(1-2):6-9. https://doi.org/10.1016/j.cca.2009.06.017

4. Haynes CA, De Jesús VR. 2016. Simultaneous quantitation of hexacosanoyl lysophosphatidylcholine, amino acids, acylcarnitines, and succinylacetone during FIA?ESI?MS/MS analysis of dried blood spot extracts for newborn screening. Clinical Biochemistry. 49(1-2):161-165. https://doi.org/10.1016/j.clinbiochem.2015.09.011

5. Chace DH, Kalas TA, Naylor EW. 2003. Use of Tandem Mass Spectrometry for Multianalyte Screening of Dried Blood Specimens from Newborns. 49(11):1797-1817. https://doi.org/10.1373/clinchem.2003.022178

6. Newborn Screening Quality Assurance Program 2020 Quality Control Report. June 1, 2020. [Internet]. Available from: https://www.cdc.gov/labstandards/pdf/nsqap/QC_Report_S1_2020-508.pdf

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