First experience with a fully automated extraction system for simultaneous on-line direct tandem mass spectrometric analysis of amino acids and (acyl-)carnitines in a newborn screening setting.

Research Article Received: 11 November 2013

Revised: 19 January 2014

Accepted: 26 January 2014

Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2014, 28, 965–973 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6856

First experience with a fully automated extraction system for simultaneous on-line direct tandem mass spectrometric analysis of amino acids and (acyl-)carnitines in a newborn screening setting Ralph Fingerhut1*, Maria Lucia Silva Polanco2, Gabriel De Jesus Silva Arevalo2 and Magdalena A. Swiderska3 1

Swiss Newborn Screening Laboratory, Children`s Research Center, University Children’s Hospital, Zurich, Switzerland INVEGEM, Instituto de Investigación de Enfermades Genéticas y Metabólicas Humanas, Campus Médico San José, Sta. Lucía Milpas Altas, Guatemala, Central America 3 CAMAG, Mutenz, Switzerland 2

RATIONALE: For Newborn Screening (NBS) programs all over the world whole blood dried on filter paper, also referred to as dried blood spots (DBS), has been the standard specimen for decades. In recent years DBS have attracted the attention of pharmaceutical companies, mostly due to the low volume of collected sample and simplified, therefore more cost-efficient, transportation requirements. However, the classical NBS workflow did not totally fulfil the needs of their studies, especially with respect to high-throughput unassisted sample processing for tandem mass spectrometric (MS/ MS) analysis. Automated on-line extraction systems for direct analysis have already been tested and proved to be suitable for these pharmaceutical applications. METHODS: The suitability of the automated CAMAG DBS-MS 500 interface for simultaneous detection of amino acids and (acyl-)carnitines has been tested together with an Acquity TQD tandem mass spectrometer from Waters and MassChrom stable isotope labelled internal standards from Chromsystems. No chromatographic sample treatment was applied; instead, the extract was directly injected into the MS/MS instrument. The feasibility of the instrumental setting for the routine newborn screening was tested on original samples coming from previously diagnosed patients. RESULTS: The performance of the automated extraction technique and its application in preliminary quantitative screening for amino acids and (acyl-)carnitines for NBS showed very promising results. Several samples from patients, each diagnosed with one of four different inborn errors of metabolism (IEM), were tested and the correlation with the conventional punch-and-elute approach was very good. CONCLUSIONS: Although the presented method still needs further optimization, our study clearly shows the possibility to use direct on-line analysis in the NBS setting. Our report on direct on-line analysis of newborn samples is a first approach in the development of a fully automated screening method for NBS analysis. With regard to the chemical properties of the analytes, the study resulted in a readily applicable screening method. Copyright © 2014 John Wiley & Sons, Ltd.

Whole blood dried on filter paper, the so-called dried blood spots (DBS), has been a standard specimen for newborn screening (NBS) since the introduction of the bacterial inhibition test for NBS for phenylketonuria (PKU) in 1963 by Robert Guthrie.[1] And, although the methods for detection of various analytes have changed over the past five decades (for review, see Fingerhut and Olgemöller[2]), punching of 3.2 mm discs from the DBS card into microtiter plates is still a standard procedure in NBS laboratories all over the world.

Rapid Commun. Mass Spectrom. 2014, 28, 965–973

Copyright © 2014 John Wiley & Sons, Ltd.

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* Correspondence to: R. Fingerhut, Swiss Newborn Screening Laboratory, University Children’s Hospital Zurich, Children`s Research Center, Steinwiesstr. 75, CH-8032 Zurich, Switzerland. E-mail: ralph.fi[email protected]

In recent years, DBS have attracted the attention of pharmaceutical companies due to the significant ethical, financial and practical benefits offered by this technique.[3,4] Despite the numerous advantages associated with the DBS methodology, one of its major drawbacks is the timeconsuming complexity of sample pre-analysis treatment when processed with the conventional ’punch-and-elute’ procedure. In response to the lack of any technology supporting more efficient and straightforward DBS sample processing, companies specialized in the development of laboratory instrumentation together with bioanalytical scientists started exploring the possibilities of systems for direct analysis in combination with mass spectrometry.[5–8] The major points in the development of this new approach were: elimination of manual DBS extraction, no requirement for a time-consuming pre-analytical sample treatment, and overall reduction of analysis cycle time per sample.

R. Fingerhut et al.

Figure 1. CAMAG DBS-MS 500 extraction module. The filter paper is fixed between the plunger and the bottom plate with inlet and outlet. This tightly squeezes a 3.2 mm circle of the dried blood spot. Extraction solvent is then pumped with a defined flow rate from one border of the circle through the whole 3.2 mm of the blood spot to the opposite border. Over the last years there has been a growing number of articles reporting successful application of various technological solutions for direct analysis in a broad spectrum of bioanalytical fields, with a major emphasis on drugs and their metabolites.[9–11] Among these, an on-line extraction has proved to be readily compatible with the needs of high-throughput sample screening.[12–15] This technique completely eliminates the tedious disc punching step, and further steps of sample treatment and liquid handling. Instead, all those complex and time-consuming operations are replaced by one continues process which, with the present fully automated solutions, can be performed with minimal human interference.

Following the development of technology for the direct analysis of DBS in the pharmaceutical industry, several laboratories reported their work on applying this approach in targeting inborn errors of metabolism (IEM).[16,17] Among these, there have been only few reports describing direct analysis through on-line DBS sample extraction coupled to tandem mass spectrometry (MS/MS), where the authors focused on quantitation of either selected (acyl-)carnitines[18] or selected amino acids.[19] However, since the first description of MS/MS as a tool for NBS,[20] the simultaneous measurement of both amino acids and acylcarnitines from a single 3.2 mm spot has been the standard procedure in expanded NBS programs. Herein, we report the first attempt in the simultaneous quantitation of amino acids and (acyl-)carnitines applying the concept of direct analysis with on-line extraction. A combination of a fully automated CAMAG DBS-MS 500 extraction system coupled directly to an electrospray ionisation tandem mass spectrometry (ESI-MS/MS) setup was used in this study. One of our major goals was to facilitate a preliminary procedure which would enable a significant reduction in sample processing time. In the newborn screening environment the high number of samples and the need for fast generation of results call for innovation and streamlining of the overall analytical process. With this study we hope to be able to contribute in the general concept of laboratory automation with special emphasis on the laboratory work for IEM and NBS.

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Figure 2. Fluidic system of the DBS-MS 500 automated platform. (a) Sample extraction process: Extraction solvent is pumped by the elution pump through the tightly squeezed circle of the dried blood spot into the sample loop. (b) Sample analysis and extraction module rinsing: After the sample loop has been filled with the extract, first the filter card is removed from the extraction module. Then the rinsing unit is positioned between the plunger and the inlet/outlet unit. Now the valve switches and while the extract is transferred with flow solvent from the LC pump to the mass spectrometer, the extraction head is simultaneously cleaned with rinsing solution.

wileyonlinelibrary.com/journal/rcm



Online analysis of amino acids and acylcarnitines in newborn screening

Figure 3. DBS-MS 500 extraction method optimization tools. (a) Internal standards can be either applied to the dried blood sample by spraying with the spray device prior to extraction, or it can be added to the extraction solution. (b) Four different IS standard solutions can be loaded simultaneously for different applications. (c) Extraction volume is variable, from as low as 5 μL. (d) Variable flow rate of the extraction volume. (e) Four different rinsing solutions can be loaded simultaneously, up to 10 washing cycles are possible, and the extraction head can be cleaned either through the inlet or through the outlet.

EXPERIMENTAL Reagents and samples Mass Chrom amino acids, acylcarnitines (non deriv.), internal standards (REF 57004), and rinsing solution (REF 57007) containing mainly acetonitrile were from Chromsystems (Gräfelfing, Germany). Heptafluorobutyric acid, acetonitrile (Chromasolv), and 2-propanol (Chromasolv) were from Sigma-Aldrich (Steinheim, Germany). Formic acid (98–100%, ACS, Reag. Ph Eur) was from Merck (Darmstadt, Germany). Dried blood control samples (low and high) were from the NeoBase non-derivatized MSMS kit (3040-0010) from PerkinElmer (Turku, Finland). Dried blood samples from patients with confirmed diagnosis were from the Swiss Newborn Screening program and deposited on Ahlstrom 226 filter paper. Instrumentation

Figure 4. Comparison of phenylalanine concentrations (in μmol/L) measured with the routine newborn screening after solvent extraction from 3.2 mm punches and on-line extraction with the CAMAG DBS-MS 500.

CAMAG DBS-MS-500


whole thickness of the filter paper (Fig. 2(a)). The device has its own built-in pump system responsible for the rinsing and the extraction fluidics. The flow rate and volume of the extraction solvent applied are optimized independently of the liquid chromatography (LC) mobile phase conditions (Fig. 3). During the extraction the sample is transferred to the sample loop. When this process is finished the internal valve of the system switches and two parallel processes take place: the sample loop is switched to the flow of the mobile phase for further LC/MS/MS analysis, and all the parts previously in contact with the sample (extraction head, capillaries) are washed by the specially designed rinsing unit (Fig. 2(b)). Multiple rinsing methods can be programmed using various numbers of rinsing cycles, volumes or solvent types. MS/MS acquisition is automatically initiated via contact closure detected by Masslynx V4.1 SCN 714 software.



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The described fully automated extraction system DBS-MS 500 is designed to handle up to 500 DBS cards. It consists of three modules: OCR (optical card and sample recognition), internal standard (IS) spraying device, and extraction head. The system is also equipped with a robotic arm, which directs the cards to each module according to the sequence previously programmed by the user. In this report we focused our studies mainly on the extraction module using the OCR as support to the sample recognition and identification of its center for the further extraction placement. The extraction head consists of an upper part, a plunger with a circular shape of 4 mm diameter, and a lower part with inlet and outlet, for the circulation of the solvent (Fig. 1). The sample is placed in the extraction module by the robotic arm and sealed between the two parts for the extraction to begin. During this process the solvent flows vertically through the

R. Fingerhut et al. Table 1. Accuracy, intra-assay and inter-assay %CV of 12 amino acids, at two different concentrations for uncharged amino acids and citrulline, and 2 different samples of the same concentration for the other amino acids

Uncharged nonpolar

Uncharged polar

Basic

Acidic

a

Target value

Accuracy [%]

Intra-assay CVa [%]

Inter-assay CVa [%]

763 1812 382 833 203 583 545 1623 454 1062 1147 2932 391 1227 8.2 8.2 101 299 55.2 55.2 35.9 35.9 30.8 30.8

96-107 105-112 101-111 107-113 104-114 111-118 96-107 101-109 102-111 109-115 86-102 103-109 95-106 103-111 69-124 80-126 79-101 97-112 32-74 36-82 60-124 60-124 84-117 74-116

4.0 3.4 4.1 3.3 4.9 3.3 4.5 3.9 5.0 3.3 5.6 4.7 4.7 4.1 12.6 10.9 7.5 6.4 17.5 18.1 13.3 13.7 7.0 9.1

8.4 7.1 7.0 5.5 8.3 7.0 8.1 6.2 6.3 6.2 13.0 10.0 10.1 8.7 56.1 37.3 27.1 13.8 96.3 79.0 59.5 59.8 28.5 27.2

Ala Ala Leu Leu Phe Phe Pro Pro Val Val Gly Gly Tyr Tyr Arg Arg Cit Cit Orn Orn Asp Asp Glu Glu

n = 10.

Table 2. Accuracy, intra-assay and inter-assay %CV of free carnitine and 12 acylcarnitines, at two different concentrations

C0 C0 C2 C2 C3 C3 C4 C4 C5 C5 C5DC C5DC C6 C6 C8 C8 C10 C10 C12 C12 C14 C14 C16 C16 C18 C18 a

Target value

Accuracy [%]

Intra-assay CVa [%]

Inter-assay CVa [%]

100 231 58.9 142.4 10.8 27.7 2.81 7.3 1.22 3.14 0.62 1.54 0.57 1.47 0.68 1.74 0.99 2.69 1.69 4.66 1.83 4.89 11.6 30.3 2.3 4.65

94–103 98–104 101–107 107–110 100–103 104–108 100–105 105–108 99–104 104–107 85–100 93–107 103–106 110–110 104–105 108–112 104–113 108–120 106–106 107–110 107–110 110–120 101–111 103–118 103–115 106–126

4.3 4.3 4.5 2.5 5.6 3.6 5.2 3.8 5.3 1.5 7.0 7.1 5.0 3.4 6.4 2.9 6.5 3.8 7.1 4.1 6.9 4.9 8.0 5.5 8.9 5.7

7.9 8.3 10.6 9.1 9.4 7.8 9.3 8.4 9.1 8.3 28.3 26.5 11.3 8.5 10.9 12.4 15.0 11.2 15.0 11.2 17.2 22.0 30.4 24.4 30.4 30.7

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n = 10.


ESI-MS/MS The Waters Acquity TQ detector, Waters 1525 μ Binary HPLC pump, and MassLynx V4.1 SCN805 software were from Waters (Milford, USA). Statistical analysis was performed with SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Quantitation of amino acids and (acyl-)carnitines from DBS Automated extraction (CAMAG DBS-MS 500) For the preparation of extraction solution, MassChrom internal standards were reconstituted according to the package insert; however, 25 mL methanol/water (9:1 v/v) containing 0.1% formic acid instead of the MassChrom extraction buffer was used for reconstitution. Amino acids and acylcarnitines were extracted from the DBS with the Table 3. Linear range, slope, and coefficient of correlation (R2)

Analyte

Slope

R2

Alanine Arginine Citrulline Leucine/Isoleucine Methionine Ornithine Phenylalanine Proline Tyrosine Valine

1.154 0.918 0.731 1.187 1.129 0.872 1.335 1.151 1.782 1.076

0.985 0.996 0.996 0.986 0.989 0.997 0.994 0.992 0.985 0.994


Linear range n [μmol/L] (total) 0 – 3045 0 – 950 0 – 1023 0 – 1020 0 – 450 0 – 1160 0 – 750 0 – 1550 0 – 850 0 – 1100

32 24 24 24 24 24 36 24 36 24



Figure 5. (a) Comparison of the amino acid profile of a normal newborn and a newborn with PKU measured with the routine newborn screening (NBS) after solvent extraction from 3.2 mm punches and on-line extraction with the CAMAG DBS-MS 500. (b) Comparison of the acylcarnitine profile of a normal newborn and a newborn with MCADD measured with the routine NBS after solvent extraction from 3.2 mm punches and on-line extraction with the CAMAG DBS-MS 500. (c) Comparison of the acylcarnitine profile of a normal newborn and a newborn with PA measured with the routine NBS after solvent extraction from 3.2 mm punches and on-line extraction with the CAMAG DBS-MS 500. (d) Comparison of the amino acid profile of a normal newborn and a newborn with MSUD measured with the routine NBS after solvent extraction from 3.2 mm punches and on-line extraction with the CAMAG DBS-MS 500. (Note: IS concentrations of leucine and valine are significantly different in the two test-kits; therefore, amino acid profiles not easily comparable between the two test-kits.)


mass spectrometer at a flow rate of 0.01 μL/min with MassChrom rinsing solution containing 0.1% formic acid and 0.01% heptafluorobutyric acid. ESI-MS/MS was run in positive ion mode with the following settings: capillary voltage, 3.50 kV; extractor voltage, 3.00 V; RF voltage, 0.10 V; source temperature, 120°C; desolvation gas temperature, 300°C; cone gas flow, 10 L/h; desolvation gas flow, 600 L/h; collision gas flow, 0.17 mL/min; cone voltage and collision energy were optimised for each analyte. For the quantitation of amino acids and acylcarnitines, the



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CAMAG DBS-MS-500 interface with 100 μL of this extraction solution at a flow rate of 50 μL/min. The extract was collected in a 100 μL sample loop integrated in the system. All connections in this configuration were made using 0.005" PEEK tubing and finger-tight fittings.When the extraction process was completed, the outlet of the extraction head of the CAMAG DBS-MS 500 interface was washed twice for 20 s with rinsing solution (acetonitrile/water/2-propanol; 4/3/3 v/v/v; containing 0.1% formic acid). In parallel the content of the sample loop was directly applied to the tandem

R. Fingerhut et al.

Figure 5. (Continued)

correction factors that are necessary to compensate for the different extraction efficiencies were determined with whole blood samples from a healthy volunteer, spiked with different amounts of the respective analytes. Punch-and-elute extraction

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The NeoBase non-derivatized MS/MS assay from PerkinElmer was used for routine analysis. 3.2 mm DBS discs were punched out of the sampling paper and transferred to a 96-well microtitetr plate containing 100 μL of the standard extraction buffer (NeoBase) with internal standards (IS) for each of the measured analytes. The extraction was performed by shaking for 45 min at 45°C; after that time the plates were placed in the CTC autosampler for injection into the tandem mass spectrometer.


RESULTS AND DISCUSSION The fully automated system for dried blood spot extraction presented here, CAMAG DBS-MS 500, proved to be very reliable from the point of view of the hardware and automation performance. There was no leakage from the clamp observed, as the clamping pressure is factory adjusted to the type of filter paper (when sampled with blood). Concerning the analytical aspects of this work several very crucial points had to be considered in the method optimisation. The key points in the final method setting were the adjustment of the flow rate, and the volume and composition of the extraction solvent. We tested two sample loop volumes: 20 and 100 μL, from which the last one gave strikingly better results when set together with 100 μL extraction volume.




Figure 5. (Continued)

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R. Fingerhut et al. Table 4. Mean difference of specific marker metabolites necessary for the detection of PKU, MSUD, MCADD, and PA

Analyte Alanine Phenylalanine Tyrosine Leucine/Isoleucine Valine Glycine Free carnitine (C0) Acetyl carnitine (C2) Propionyl carnitine (C3) Hexanoyl carnitine (C6) Octanoyl carnitine (C8) Decanoyl carnitine (C10) Decenoyl carnitine (C10:1) Hexadecanoyl carnitine (C16)

Mean difference [%]

Range [μmol/L]

n (total)

3.7 3.6 5.5 –1.1 –8.8 3.9 8.1 –5.0 7.4

78–334 22–977 19–136 81–413 29–351 132–1520 6.8–41 2.8–29 0.2–31

25 25 25 25 25 25 25 25 25

3.2

0.2–0.7*

4

9.8

0.8–2.6*

4

8.9

0.1–0.2*

4

8.6

0.1–0.4*

4

–1.7

0.4–1.7

25

*All values below 0.1 μmol/L were equally low with both methods; however, calculation of relative difference would lead to erroneously high values.

3.3–5.6%. Inter-assay CVs were between 5.5–8.4% for the uncharged nonpolar amino acids, and 8.7–13.0% for the uncharged polar amino acids. From the basic and acidic amino acids only citrulline was spiked and showed still reasonable CVs. All spiked acylcarnitines showed recoveries between 85–126%. Intra-assay CVs were between 2.5–8.9% for all acylcarnitines. However, inter-assay CVs showed increasing values from free carnitine and short-chain acylcarnitines, over medium-chain acylcarnitines, to long-chain acylcarnitines and glutarylcarnitine. For a subset of analytes also linearity was calculated (Table 3, Fig. 4). For this purpose blood of a healthy adult volunteer was spiked with six different concentrations of the ten different amino acids (see Table 3), resulting in sevensamples with different analyte concentrations. We also measured various samples from newborns with a confirmed IEM (PKU, Medium-Chain Acyl-CoA Dehydrogenase Deficiency [MCADD], propionic acidaemia [PA], maple syrup urine disease [MSUD]), and compared the results with those measured from the same specimen with the routine newborn screening method after extraction from 3.2 mm punches (Figs. 5(a)–5(d)). In total we had 28 measurements from 16 different samples. The mean difference between the results of all respective marker metabolites, necessary for the detection of the corresponding IEMs, from routine NBS and on-line direct MS/MS measurement were all within ±10% (Table 4). The variation of the results between multiple measurements of the same sample with the routine NBS method has approximately the same magnitude as the variation between the two different methods (data not shown). With the DBS-MS 500 on-line extraction the first results are available at least 1.5 h earlier than with the routine newborn screening method and with one-third of the manpower, 20 min instead of 60 min is needed to process 90 samples (Fig. 6). Overall the collected data suggest that further optimization of the composition of extraction solvent and the necessary extraction volume is still required. In the near future the software integration, which will enable easier communication between the DBS-MS 500 and the MS/ MS system, and additional studies on the possibilities of IS spraying application will complete the study.

CONCLUSIONS Figure 6. Comparison of timescale and workload for the routine newborn screening after solvent extraction from 3.2 mm punches and on-line extraction with the CAMAG DBS-MS 500.

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We noticed that the extraction with the NeoBase nonderivatized kit extraction buffer resulted in high ion suppression and poor signal, preventing quantitation of analytes with the CAMAG DBS-500 on-line extraction system. The addition of heptafluorobutyric acid and application of a 2 μm in-line stainless frit on the way to mass spectrometer drastically improved the results. Tables 1 and 2 present the accuracy and precision for 12 amino acids and 13 acylcarnitines, respectively. Accuracy was between 86–118 % for the uncharged amino acid, with intra-assay coefficients of variance (CVs) between


The data we present is to our knowledge the first report of a fully automated on-line screening method for NBS analysis. All previous work has been focused on specific analyte species. However, if the method is to be applicable for NBS, the whole panel of amino acids and acylcarnitines have to be covered. Since the chemical properties of the analytes cover the whole spectrum from easily soluble free carnitine to hydrophobic long-chain acylcarnitines, and from nonpolar uncharged amino acids to basic or acidic amino acids, the choice of a suitable extraction solution is a big challenge. So far we could show that the described method is already capable of detecting certain IEM with the same accuracy as with the routine NBS method. This is especially true for free carnitine, short-, and medium-chain acylcarnitines, as well as for uncharged nonpolar amino acids and to a slightly lesser extent also for the uncharged polar amino acids. With the current setting, we have already proven that PKU, MSUD,



Online analysis of amino acids and acylcarnitines in newborn screening MCADD, and PA/MMA (methylmalonic aciduria) can be detected with the on-line extraction method, and from the results of control samples also isovaleric acidaemia, with isovaleryl-carnitine (C5) as the primary marker should also be easily detectable. For the other analytes and disorders that are already part of the newborn screening panel in many countries, we are searching for more suitable extraction conditions. This is part of an ongoing study which should result in extraction properties that are suitable for the whole panel of extended NBS. For the dietary monitoring of patients with PKU, DBS are already the standard specimen. However, with on-line extraction coupled to MS/MS, the analysis time can be shortened significantly. In the routine setting, if samples are sent by regular post, results are ready the next working day. With on-line extraction, results can be available within 10–15 min after the samples arrive in the laboratory. In a clinical setting like in the University Children`s Hospital in Zurich, patients could have blood drawn, once they come to the outpatient clinic, and actual phenylalanine levels would be available during the consultation with the paediatrician or the dietician.

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[8] P. Abu-Rabie. Direct analysis of DBS: emerging and desirable technologies. Bioanalysis 2011, 3, 1675. [9] J. M. Wiseman, C. A. Evans, C. L. Bowen, J. H. Kennedy. Direct analysis of dried blood spots utilizing desorption electrospray ionization (DESI) mass spectrometry. Analyst 2010, 135, 720. [10] J. Liu, H. Wang, N. E. Manicke, J.-M. Lin, R. Graham Cooks, Z. Ouyang. Development, characterization, and application of paper spray ionization. Anal. Chem. 2010, 82, 2463. [11] E. Crawford, J. Gordon, J.-T. Wu, B. Musselman, R. Liu, S. Yu. Direct analysis in real time coupled with dried spot sampling for bioanalysis in a drug-discovery setting. Bioanalysis 2011, 3, 1217. [12] P. Abu-Rabie, N. Spooner. Direct quantitative bioanalysis of drugs in dried blood spot samples using a thin-layer chromatography mass spectrometer interface. Anal. Chem. 2009, 81, 10275. [13] P. Abu-Rabie, N. Spooner. Dried matrix spot direct analysis: evaluating the robustness of a direct elution technique for use in quantitative bioanalysis. Bioanalysis 2011, 3, 2769. [14] J. Déglon, A. Thomas, A. Cataldo, P. Mangin, C. Staub. Online desorption of dried blood spot: A novel approach for the direct LC/MS analysis of micro-whole blood samples. J. Pharm. Biomed. Anal. 2009, 49, 1034. [15] J. Déglon, A. Thomas, Y. Daali, E. Lauer, C. Samer, J. Desmeules, P. Dayer, P. Mangin, C. Staub. Automated system for on-line desorption of dried blood spots applied to LC/MS/MS pharmacokinetic study of flurbiprofen and its metabolite. J. Pharm. Biomed. Anal. 2011, 54, 359. [16] C. Wang, H. Zhu, Z. Cai, F. Song, Z. Liu, S. Liu. Newborn screening of phenylketonuria using direct analysis in real time (DART) mass spectrometry. Anal. Bioanal. Chem. 2013, 405, 3159. [17] P. Britz-McKibbin. Expanded newborn screening of inborn errors of metabolism by capillary electrophoresiselectrospray ionization-mass spectrometry (CE-ESI-MS). Methods Mol. Biol. 2013, 919, 43. [18] J. W. Thompson, H. Zhang, P. Smith, S. Hillman, M. A. Moseley, D. S. Millington. Extraction and analysis of carnitine and acylcarnitines by electrospray ionization tandem mass spectrometry directly from dried blood and plasma spots using a novel autosampler. Rapid Commun. Mass Spectrom. 2012, 26, 2548. [19] J. H. Miller 4th, P. A. Poston, H. T. Karnes. Direct analysis of dried blood spots by in-line desorption combined with high-resolution chromatography and mass spectrometry for quantification of maple syrup urine disease biomarkers leucine and isoleucine. Anal. Bioanal. Chem. 2011, 400, 237. [20] D. H. Chace, D. S. Millington, N. Terada, S. G. Kahler, C. R. Roe, L. F. Hofman. Rapid diagnosis of phenylketonuria by quantitative analysis of phenylalanine and tyrosine in neonatal blood spots by tandem mass spectrometry. Clin. Chem. 1993, 39, 66.

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