**1.6 Nonketotic hyperglycemia**

Nonketotic hyperglycemia (NKH) is a severe disorder of glycine metabolism. Glycine is catabolized through the four-peptide cleavage complex. P-protein, a pyridoxal phosphate-containing protein, T-protein, a protein required for the tetrahydrofolate-dependent reaction, H-protein, a protein that carries the aminomethyl intermediate and then hydrogen through the prosthetic lipoyl moiety, and L-protein, a lipoamide dehydrogenase. The disorder is so severe, that most of the affected individuals die within few months of life or survive with significant intellectual disabilities. Main laboratory findings in NKH is plasma and CSF elevated glycine.


#### **Table 5.**

*Laboratory findings in renal aminoacidurias.*


#### **Table 6.**

*Laboratory findings in NKH.*

#### **1.7 Renal aminoacidurias**

Renal aminoacidurias are disorders have inherited defects that affect renal tubular reabsorption process. Thus these disorders are characterized by abnormal urinary amino acids.

#### **2. Newborn screening**

Early diagnosis may prevent serious implications of inborn errors of metabolism, including amino acids disorders and significantly decrease morbidity and mortality. Newborn screening is a public health program that facilities early diagnosis by identifying neonates with potential treatable inborn errors of metabolism at the very early stages of their lives [13, 14]. This practice helps to manage the disease even for neonates that do not have evident symptoms in the first days of their lives. Amino acids analysis has always been an important part of the newborn screening. The first PKU screening bacterial inhibition assay was invented by Robert Guthrie in the early '60s [15]. Since that time, screening for IEMs is performed worldwide. In the United States, newborn screening is a state-mandated public health program ensuring that all newborns are screened for certain inherited conditions at birth. The panel of screening conditions varies from state to state. The advisory committee on heritable disorders in newborns and children advises the Secretary of Health and human services uniform screening panel, which currently consist of 34 core disorders and 26 secondary disorders. The recommended panel includes multiple amino acids related disorders (**Table 7**).


#### **Table 7.**

*List of amino acids disorders that are recommended by the Secretary of the Department of Health and Human Services (HHS) as a part of state universal newborn screening (NBS) program effective July 2018.*

#### **3. Quantitative amino acids analysis**

Quantitative amino acids analysis is an important tool for diagnosis of amino acids disorders and nutritional monitoring of individuals with already established diagnosis. Amino acids can be detected in most biological fluids, however, the most common

**121**

mask diagnostic findings.

*Amino Acids Profiling for the Diagnosis of Metabolic Disorders*

fluids for inborn errors of metabolism diagnostics and monitoring are blood, plasma, and urine. In some cases, cerebrospinal fluid (CSF) amino acid levels are also diagnostic (**Table 5**). Although each disorder is biochemically characterized by abnormal levels of a single or a few amino acids, quantitative a non-screening analysis, and interpretation is not restricted to those metabolites and consist from a panel of nearly 40 amino acids and specific ratios. For example, along with plasma phenylalanine level, it is important also to assess plasma phenylalanine/tyrosine ratio that can be used to differentiate between PKU and non-PKU hyperphenylalaninemia [16].

The different chemical characteristics, a wide range of normal physiological levels [17–19], age groups variability and other factors detailed below represent a significant analytical challenge for the amino acid analysis. Diet is one of the significant factors that can highly affect amino acids levels [20, 21]. For example, meat and poultry consumption leads to increased excretion of β alanine and 1-methylhistidine. Thus blood collection intended for amino acids analysis is recommended after overnight fasting. Other factors such as urinary bacterial contamination can significantly alter urinary amino acids profile [22]. Some drugs interfere with amino acids metabolism [23] or cause signal artifacts. Valproic acid, for example, can cause an increase in plasma glycine. Anticoagulants used during sample collection also can contain interfering constituents [24]. For example, blood collection tubes containing sodium bisulfate in addition to heparin can yield a peak of S-sulfocysteine, falsely suggesting sulfite oxidase deficiency. Ethylenediaminetetraacetic acid (EDTA) additive in collection tube can produce ninhydrin-positive peaks, therefore lithium-heparin coated tubes are strongly preferred for the blood collection. An additional interfering factor to the amino acid analysis is a hemolysis as it may lead to the decrease of arginine with simultaneous increase of ornithine due to red blood cells arginase activity, and an increase in taurine that released from leukocytes and platelets. Serum is usually not a choice for the amino acids analysis, because blood needs to clot at room temperature during which asparagine is converted to aspartic acid and glutamine to glutamic acid. For the urine analysis, a 24-h urine collection is preferred, alternatively, an overnight collection can be sufficient for the diagnostic purposes. In order to avoid

Overall, during a prolong sample storage glutamine and asparagine decrease whereas glutamic and aspartic acids increase simultaneously. Additional markers of prolong storage are an increase of ethanolamine derived from phosphoethanol-

When cerebrospinal fluid is used for the analysis, it must be not contaminated with blood, as it leads to the nonspecific increase of multiple amino acids and can

Quantitative amino acids analysis implies in a variety of nonclinical fields such as biomedical research, bioengineering, food science, and agriculture. Multiple analytical methods have been developed over the years, however, some of these methods are not cost effective and labor intensive and thus are not applicable in clinical settings. The aim of next paragraphs is to describe the most common and

In early 50s, diagnostic quantitative amino acid analysis became feasible with Moore and Stein publication on plasma amino acids separation with polystyrene

*DOI: http://dx.doi.org/10.5772/intechopen.84672*

**3.1 Factors affecting amino acid analysis**

artifacts, no preservatives are added to the urine sample.

widely used platforms in laboratory medicine field.

amine decomposition, increased tryptophan, GABA and taurine.

**3.2 Ion exchange chromatography coupled with optical detection**

*Amino Acids Profiling for the Diagnosis of Metabolic Disorders DOI: http://dx.doi.org/10.5772/intechopen.84672*

*Biochemical Testing - Clinical correlation and Diagnosis*

**involved**

Gly (B, CSF) high

Renal aminoacidurias are disorders have inherited defects that affect renal tubular reabsorption process. Thus these disorders are characterized by abnormal

system

**Enzyme/transport defect Additional biomarkers**

Increased CSF/plasma

Gly ratio

Mutations in Gly cleavage

Early diagnosis may prevent serious implications of inborn errors of metabolism, including amino acids disorders and significantly decrease morbidity and mortality. Newborn screening is a public health program that facilities early diagnosis by identifying neonates with potential treatable inborn errors of metabolism at the very early stages of their lives [13, 14]. This practice helps to manage the disease even for neonates that do not have evident symptoms in the first days of their lives. Amino acids analysis has always been an important part of the newborn screening. The first PKU screening bacterial inhibition assay was invented by Robert Guthrie in the early '60s [15]. Since that time, screening for IEMs is performed worldwide. In the United States, newborn screening is a state-mandated public health program ensuring that all newborns are screened for certain inherited conditions at birth. The panel of screening conditions varies from state to state. The advisory committee on heritable disorders in newborns and children advises the Secretary of Health and human services uniform screening panel, which currently consist of 34 core disorders and 26 secondary disorders. The recommended panel includes multiple amino acids related disorders (**Table 7**).

Quantitative amino acids analysis is an important tool for diagnosis of amino acids disorders and nutritional monitoring of individuals with already established diagnosis. Amino acids can be detected in most biological fluids, however, the most common

**Argininosuccinate aciduria, citrullinemia type I homocystinuria (cystathionine-β-synthase), maple syrup urine disease, phenylketonuria/**

Nonketotic hyperglycinemia (NKH), prolinemia, hyperammonemia/ornithinemia/

**hyperphenylalaninemia, tyrosinemia I**

*Services (HHS) as a part of state universal newborn screening (NBS) program effective July 2018.*

Secondary Defects of biopterin cofactor biosynthesis and regeneration, citrullinemia II, hypermethioninemia, tyrosinemia II, tyrosinemia III

*List of amino acids disorders that are recommended by the Secretary of the Department of Health and Human* 

citrullinemia (HHH)

**1.7 Renal aminoacidurias**

Nonketotic hyperglycemia

*B, blood; CSF, cerebrospinal fluid.*

*Laboratory findings in NKH.*

(NKH)

**Table 6.**

**Disorder of glycine metabolism**

**Disorder name Amino acid** 

urinary amino acids.

**Recommended uniform screening panel (RUSP)**

Additional non-RUSP conditions

**2. Newborn screening**

**120**

**Table 7.**

**3. Quantitative amino acids analysis**

fluids for inborn errors of metabolism diagnostics and monitoring are blood, plasma, and urine. In some cases, cerebrospinal fluid (CSF) amino acid levels are also diagnostic (**Table 5**). Although each disorder is biochemically characterized by abnormal levels of a single or a few amino acids, quantitative a non-screening analysis, and interpretation is not restricted to those metabolites and consist from a panel of nearly 40 amino acids and specific ratios. For example, along with plasma phenylalanine level, it is important also to assess plasma phenylalanine/tyrosine ratio that can be used to differentiate between PKU and non-PKU hyperphenylalaninemia [16].

#### **3.1 Factors affecting amino acid analysis**

The different chemical characteristics, a wide range of normal physiological levels [17–19], age groups variability and other factors detailed below represent a significant analytical challenge for the amino acid analysis. Diet is one of the significant factors that can highly affect amino acids levels [20, 21]. For example, meat and poultry consumption leads to increased excretion of β alanine and 1-methylhistidine. Thus blood collection intended for amino acids analysis is recommended after overnight fasting. Other factors such as urinary bacterial contamination can significantly alter urinary amino acids profile [22]. Some drugs interfere with amino acids metabolism [23] or cause signal artifacts. Valproic acid, for example, can cause an increase in plasma glycine. Anticoagulants used during sample collection also can contain interfering constituents [24]. For example, blood collection tubes containing sodium bisulfate in addition to heparin can yield a peak of S-sulfocysteine, falsely suggesting sulfite oxidase deficiency. Ethylenediaminetetraacetic acid (EDTA) additive in collection tube can produce ninhydrin-positive peaks, therefore lithium-heparin coated tubes are strongly preferred for the blood collection. An additional interfering factor to the amino acid analysis is a hemolysis as it may lead to the decrease of arginine with simultaneous increase of ornithine due to red blood cells arginase activity, and an increase in taurine that released from leukocytes and platelets. Serum is usually not a choice for the amino acids analysis, because blood needs to clot at room temperature during which asparagine is converted to aspartic acid and glutamine to glutamic acid.

For the urine analysis, a 24-h urine collection is preferred, alternatively, an overnight collection can be sufficient for the diagnostic purposes. In order to avoid artifacts, no preservatives are added to the urine sample.

Overall, during a prolong sample storage glutamine and asparagine decrease whereas glutamic and aspartic acids increase simultaneously. Additional markers of prolong storage are an increase of ethanolamine derived from phosphoethanolamine decomposition, increased tryptophan, GABA and taurine.

When cerebrospinal fluid is used for the analysis, it must be not contaminated with blood, as it leads to the nonspecific increase of multiple amino acids and can mask diagnostic findings.

Quantitative amino acids analysis implies in a variety of nonclinical fields such as biomedical research, bioengineering, food science, and agriculture. Multiple analytical methods have been developed over the years, however, some of these methods are not cost effective and labor intensive and thus are not applicable in clinical settings. The aim of next paragraphs is to describe the most common and widely used platforms in laboratory medicine field.

#### **3.2 Ion exchange chromatography coupled with optical detection**

In early 50s, diagnostic quantitative amino acid analysis became feasible with Moore and Stein publication on plasma amino acids separation with polystyrene

resin column [25] and the subsequent automatization of the technique [26]. The principle, called ion exchange chromatography (IEC) with a post-column derivatization, for a long time remained a gold standard for the clinical amino acids analysis. Nowadays, despite the methodological advancements, the ion exchange chromatography using a lithium buffer system, followed the post-column derivatization with ninhydrin and UV detection is still widely used in clinical setting.

Standard sample preparation for IEC amino acid analysis involves deproteinization with 35% (w/v) sulfosalicylic acid (SSA) added to the biological fluid. It is recommended to use one volume of SSA to 10 volumes of plasma. A fixed amount of non-physiological amino acid as an internal standard is added to all samples. Commonly used internal standards are d-glucosaminic acid, S-2-aminoethyl-1-cysteine, norvaline, and norleucine, however, norleucine can interfere with argininosuccinic acid peak. After a short incubation, centrifugation and filtration, the sample is ready for the injection and separation.

In IEC, the separation is driven by the ionic interactions between the amino acid and functional ligands linked to the stationary phase of the column. The chromatographic column is filled with negatively charged resins. The sample is loaded on the column in low acidic pH and at this point, all amino acids bear a positive charge and strongly interact with the column. Manipulation with a lithium buffer composition during the run alters pH and salt composition, and as a result, there is a change in amino acid charge status (**Figure 5**). As the isoelectric point is reached amino acid is not charged anymore and has weak interactions with the charged column.

The complex separation of multiple amino acids is achieved based on ionic interactions strength. Amino acids with the weakest ionic interactions to the column start to elute first. After column elution, amino acids are mixed with a post-column reagent and are optically detected. The most common and wellestablished post-column derivatization is reaction with ninhydrin that produces a purple Ruhemann's chromophore (*λ*max = 570 nm, **Figure 6**) for α-amino acids and yellow product with secondary amines (*λ*max = 440 nm) for such as proline and hydroxyproline [27].

The absorbance intensity of the produced colorful analyte originated from every eluted amino acid is proportional to the amino acid's concentration in the examined biological fluid. Despite the fact that IEC amino acids technique is highly reproducible with a good linearity over a broad range, it suffers from a long run time for the full amino acids profile (about 150 min), and a lack of specificity as amino acids identification is based solely on retention time. Furthermore, co-elution of some amino acids on standard IEC method is

**123**

**Figure 7.**

*AQC reaction with amino acid.*

*Amino Acids Profiling for the Diagnosis of Metabolic Disorders*

*Ninhydrin reaction with amino acid to produce Ruhemann's purple.*

observed. For example, homocitrulline co-elutes with methionine and make challenging HHH syndrome. Moreover, allo-isoleucine, a diagnostic market for MSUD co-elutes with cystathionine. Argininosuccinic acid that accumulates in patients with argininosuccinate lyase deficiency has the same retention time as leucine. Additional drawback of the methodology is a limited stability of ninhydrin (recommended storage of the working solution ≤1 month) which adds up to

In recent years, reverse-phase high-performance liquid chromatography (RP-HPLC) and ultra-high performance liquid chromatography (UPLC) methods emerged as an alternative to the ion exchange chromatography. In RP-HPLC methods, the separation is based on hydrophobic interactions between the analyzed amino acid in the mobile phase and the immobilized hydrophobic ligands attached to the nonpolar column stationary phase. RP-HPLC offers a great resolution of very closely related molecules under a wide range of chromatographic conditions. For the optical detection, derivatization with o-phthalaldehyde (OPA) can be used as a pre-column or a post-column reaction. During the reaction, in the presence of thiol such as 2-mercaptoethanol, a stable fluorescent product is produced and can be detected by fluorimetry (excitation 340 nm and emission 410 nm) or UV (340 nm) [28, 29]. Although reproducible and automated [30], OPA derivatization method is not a good choice for proline/hydroxyproline and sulfur-containing amino acids detection. Alternative reagents for RP-HPLC with pre-column derivatization are phenylisothiocyanate (PITC, Pico-Tag commercialized by Waters) [31], dimethylamino-azobenzenesulfonyl-chloride (DABS-Cl) [32] and 9-fluorenylmethylchloroformate

More advanced UPLC systems employ a small particle size (typically 1.7 μM) and a high pH range stable columns. These systems use less solvent and are operated in a high pressure which allows an excellent resolution achieved in a short time frame and thus potentially decreases turnaround time per sample. Narayan et al. analyzed 170 patient samples by pre-column 6-aminoquinolyl-*N*-hydroxysuccinimidyl carbamate (AQC) derivatization (**Figure 7**) followed by reverse phase UPLC [34] and compared amino acids data to the traditional amino acids analyzer operated through ion

*DOI: http://dx.doi.org/10.5772/intechopen.84672*

the cost of the analysis.

**Figure 6.**

(FMOC-Cl) [33].

**3.3 RP-HPLC and RP-UPLC techniques**

**Figure 5.** *Aspartic acid charge in different pH.*

*Amino Acids Profiling for the Diagnosis of Metabolic Disorders DOI: http://dx.doi.org/10.5772/intechopen.84672*

**Figure 6.**

*Biochemical Testing - Clinical correlation and Diagnosis*

the sample is ready for the injection and separation.

setting.

hydroxyproline [27].

resin column [25] and the subsequent automatization of the technique [26]. The principle, called ion exchange chromatography (IEC) with a post-column derivatization, for a long time remained a gold standard for the clinical amino acids analysis. Nowadays, despite the methodological advancements, the ion exchange chromatography using a lithium buffer system, followed the post-column derivatization with ninhydrin and UV detection is still widely used in clinical

Standard sample preparation for IEC amino acid analysis involves deproteinization with 35% (w/v) sulfosalicylic acid (SSA) added to the biological fluid. It is recommended to use one volume of SSA to 10 volumes of plasma. A fixed amount of non-physiological amino acid as an internal standard is added to all samples. Commonly used internal standards are d-glucosaminic acid, S-2-aminoethyl-1-cysteine, norvaline, and norleucine, however, norleucine can interfere with argininosuccinic acid peak. After a short incubation, centrifugation and filtration,

In IEC, the separation is driven by the ionic interactions between the amino acid and functional ligands linked to the stationary phase of the column. The chromatographic column is filled with negatively charged resins. The sample is loaded on the column in low acidic pH and at this point, all amino acids bear a positive charge and strongly interact with the column. Manipulation with a lithium buffer composition during the run alters pH and salt composition, and as a result, there is a change in amino acid charge status (**Figure 5**). As the isoelectric point is reached amino acid is

not charged anymore and has weak interactions with the charged column.

The complex separation of multiple amino acids is achieved based on ionic interactions strength. Amino acids with the weakest ionic interactions to the column start to elute first. After column elution, amino acids are mixed with a post-column reagent and are optically detected. The most common and wellestablished post-column derivatization is reaction with ninhydrin that produces a purple Ruhemann's chromophore (*λ*max = 570 nm, **Figure 6**) for α-amino acids and yellow product with secondary amines (*λ*max = 440 nm) for such as proline and

The absorbance intensity of the produced colorful analyte originated from every eluted amino acid is proportional to the amino acid's concentration in the examined biological fluid. Despite the fact that IEC amino acids technique is highly reproducible with a good linearity over a broad range, it suffers from a long run time for the full amino acids profile (about 150 min), and a lack of specificity as amino acids identification is based solely on retention time. Furthermore, co-elution of some amino acids on standard IEC method is

**122**

**Figure 5.**

*Aspartic acid charge in different pH.*

*Ninhydrin reaction with amino acid to produce Ruhemann's purple.*

observed. For example, homocitrulline co-elutes with methionine and make challenging HHH syndrome. Moreover, allo-isoleucine, a diagnostic market for MSUD co-elutes with cystathionine. Argininosuccinic acid that accumulates in patients with argininosuccinate lyase deficiency has the same retention time as leucine. Additional drawback of the methodology is a limited stability of ninhydrin (recommended storage of the working solution ≤1 month) which adds up to the cost of the analysis.

#### **3.3 RP-HPLC and RP-UPLC techniques**

In recent years, reverse-phase high-performance liquid chromatography (RP-HPLC) and ultra-high performance liquid chromatography (UPLC) methods emerged as an alternative to the ion exchange chromatography. In RP-HPLC methods, the separation is based on hydrophobic interactions between the analyzed amino acid in the mobile phase and the immobilized hydrophobic ligands attached to the nonpolar column stationary phase. RP-HPLC offers a great resolution of very closely related molecules under a wide range of chromatographic conditions. For the optical detection, derivatization with o-phthalaldehyde (OPA) can be used as a pre-column or a post-column reaction. During the reaction, in the presence of thiol such as 2-mercaptoethanol, a stable fluorescent product is produced and can be detected by fluorimetry (excitation 340 nm and emission 410 nm) or UV (340 nm) [28, 29]. Although reproducible and automated [30], OPA derivatization method is not a good choice for proline/hydroxyproline and sulfur-containing amino acids detection. Alternative reagents for RP-HPLC with pre-column derivatization are phenylisothiocyanate (PITC, Pico-Tag commercialized by Waters) [31], dimethylamino-azobenzenesulfonyl-chloride (DABS-Cl) [32] and 9-fluorenylmethylchloroformate (FMOC-Cl) [33].

More advanced UPLC systems employ a small particle size (typically 1.7 μM) and a high pH range stable columns. These systems use less solvent and are operated in a high pressure which allows an excellent resolution achieved in a short time frame and thus potentially decreases turnaround time per sample. Narayan et al. analyzed 170 patient samples by pre-column 6-aminoquinolyl-*N*-hydroxysuccinimidyl carbamate (AQC) derivatization (**Figure 7**) followed by reverse phase UPLC [34] and compared amino acids data to the traditional amino acids analyzer operated through ion

**Figure 7.** *AQC reaction with amino acid.*

exchange chromatography method. The study found that UPLC method is comparable to the reference IEC and thus adaptable to the clinical laboratory.

Peake et al. developed a modified RP-UPLC method and achieved a better resolution for tyrosine, glycine, arginine and homocitrulline peaks [35]. The improved method also provides enhanced resolution to separate ornithine from mesocystathionine. There is a high clinical significance to accurate ornithine analysis as ornithine's levels are diagnostic for hyperornithinemia-hyperammonemiahomocitrullinemia (HHH) syndrome. The developed UPLC method has several advantages. Due to the short analysis time, it is feasible to include calibration prior to the analysis of urgent samples with a special turnaround times. Overall, RP-UPLC decreases turnaround time per sample, however, commercial kit components have a very limited shelf life and thus the method is not cost effective for clinical laboratories with a small samples volume.

Overall, ion exchange chromatography, RP-HPLC, and RP-UPLC techniques have a good reproducibility and a high sensitivity in the low picomole range, however, they all are carried out with optical detection. The main drawback of this type of detection is a lack of specificity as amino acids identification is solely based on the retention time. This can potentially cause to the false findings. For example, in a standard ion exchange chromatography method, ampicillin and amoxicillin co-elute with phenylalanine and it can be reported as falsely elevated.

#### **3.4 Flow infusion tandem mass spectrometry (FIA-MS/MS)**

More recently, developments and advancements in mass spectrometry field led to the inclusion of tandem mass spectrometry (MS/MS) as an alternative high throughput and specific technique for the amino acids analysis. It is also feasible to separate amino acids by liquid chromatography prior to the mass spectrometry analysis, however, it is time-consuming in clinical settings. Instead, tandem mass spectrometry scans are used for the high throughput, cost-effective amino acids analysis. It has to be noted, that FIA-MS/MS is a screening analysis that widely implemented through the newborn screening initiative.

For the newborn screening, blood samples are typically collected on filter paper and a defined size (typically 3 mm) disks are punched out of the paper and are extracted. The early assays required derivatization by butylation (**Figure 8**) in order to improve detection limits and minimize ion suppression effects in a complex biological matrix. Currently, to increase a throughput, some clinical laboratories skip on the derivatization step. Extracted and derivatized samples are directly introduced by injection to the mass spectrometer instrument with no chromatographic separation. Usually, 5–10 μl of a sample is injected into a flowing solvent at a very low (20–50 μl) flow rate. All screened amino acids (**Table 8**) are eluting at the same time whereas a typical run time is 1.5–2 min per sample. Every analyzed amino acid is assayed with the corresponding stable isotopic labeled standard.

The isotopic-labeled standards are closely related to the structure of the analyzed amino acids and have similar physicochemical properties to the target amino acids, but can be distinguished by mass spectrometry as they have a different

**125**

**Figure 9.**

*amino acids) passing through Q2 remains constant.*

**Table 8.**

*labeled internal standards.*

*Amino Acids Profiling for the Diagnosis of Metabolic Disorders*

Alanine 146.1 <sup>2</sup>

Arginine 231.2 13C,2

Aspartic acid 246.2 <sup>2</sup>

Citrulline 232.2 <sup>2</sup>

Leucine/isoleucine 188.2 <sup>2</sup>

Methionine 206.2 <sup>2</sup>

Ornithine 189.2 <sup>2</sup>

Valine 174.2 <sup>2</sup>

Glutamic acid 260.2 <sup>2</sup>

mass to charge ratio (*m*/*z*) (**Table 8**). They are added at a known quantity, and the response of each analyzed amino acid is normalized by the response of the matching internal standard. This type of normalization reduces a systematic error due to the poor recovery and decreases multiple matrix effects. The inclusion of internal standards also corrects a batch to batch variability due to the sample preparation

*Amino acids analyzed by FIA-MS/MS for the standard newborn screening panel and their stable isotopic* 

**Target amino acid** *m***/***z* **Internal standard** *m***/***z*

Glycine 132.1 15N,13C glycine 134.1

Phenylalanine 222.2 13C6 phenylalanine 228.2 Tyrosine 238.2 13C6 tyrosine 244.2

H4 alanine 150.1

H4 arginine 236.2

H3 aspartic acid 249.2

H2 citrulline 234.2

h3 leucine 191.2

H3 methionine 209.2

H2 ornithine 191.2

H8 valine 182.2

H3 glutamic acid 263.2

The tandem mass spectrometer has five basic components: the ion source where all molecules are a subject to the soft ionization, a mass analyzer that separates analytes based on their mass to charge ratio (Q1), a collision cell where molecular ions encounter an inert gas and undergo fragmentation (Q2), a second mass analyzer to separate fragments produced in the collision cell (Q3), and a detector. In collision cell, most of the screened butylated α-amino acids form a common and a very specific fragment of 102 Da (**Figure 9**). The tandem mass spectrometer then can be set to scan a constant mass difference of 102 Da and to produce a spectrum of the molecular ions derived from those amino acids that lost 102 Da in the collision cell (Q2) (**Figure 9**). Butylated amino acids with a basic side chains such as ornithine, citrulline loose ammonia and butyl formate in the

*Schematic presentation of tandem mass spectrometer. Phenylalanine (as butyl ester) looses 106 Da in the collision cell. When mass spectrometer operates in neutral loss scanning mode, it scans Q1 and Q3 in a synchronized manner. The mass difference of 102 Da (corresponds to a neutral fragment common to the most* 

and overall raises the accuracy and precision of the assay.

*DOI: http://dx.doi.org/10.5772/intechopen.84672*

**Figure 8.** *Derivatization of alanine with n-butanol.*


#### *Amino Acids Profiling for the Diagnosis of Metabolic Disorders DOI: http://dx.doi.org/10.5772/intechopen.84672*

#### **Table 8.**

*Biochemical Testing - Clinical correlation and Diagnosis*

clinical laboratories with a small samples volume.

with phenylalanine and it can be reported as falsely elevated.

**3.4 Flow infusion tandem mass spectrometry (FIA-MS/MS)**

implemented through the newborn screening initiative.

exchange chromatography method. The study found that UPLC method is compa-

Peake et al. developed a modified RP-UPLC method and achieved a better resolution for tyrosine, glycine, arginine and homocitrulline peaks [35]. The improved method also provides enhanced resolution to separate ornithine from mesocystathionine. There is a high clinical significance to accurate ornithine analysis as ornithine's levels are diagnostic for hyperornithinemia-hyperammonemiahomocitrullinemia (HHH) syndrome. The developed UPLC method has several advantages. Due to the short analysis time, it is feasible to include calibration prior to the analysis of urgent samples with a special turnaround times. Overall, RP-UPLC decreases turnaround time per sample, however, commercial kit components have a very limited shelf life and thus the method is not cost effective for

Overall, ion exchange chromatography, RP-HPLC, and RP-UPLC techniques have a good reproducibility and a high sensitivity in the low picomole range, however, they all are carried out with optical detection. The main drawback of this type of detection is a lack of specificity as amino acids identification is solely based on the retention time. This can potentially cause to the false findings. For example, in a standard ion exchange chromatography method, ampicillin and amoxicillin co-elute

More recently, developments and advancements in mass spectrometry field led to the inclusion of tandem mass spectrometry (MS/MS) as an alternative high throughput and specific technique for the amino acids analysis. It is also feasible to separate amino acids by liquid chromatography prior to the mass spectrometry analysis, however, it is time-consuming in clinical settings. Instead, tandem mass spectrometry scans are used for the high throughput, cost-effective amino acids analysis. It has to be noted, that FIA-MS/MS is a screening analysis that widely

For the newborn screening, blood samples are typically collected on filter paper

and a defined size (typically 3 mm) disks are punched out of the paper and are extracted. The early assays required derivatization by butylation (**Figure 8**) in order to improve detection limits and minimize ion suppression effects in a complex biological matrix. Currently, to increase a throughput, some clinical laboratories skip on the derivatization step. Extracted and derivatized samples are directly introduced by injection to the mass spectrometer instrument with no chromatographic separation. Usually, 5–10 μl of a sample is injected into a flowing solvent at a very low (20–50 μl) flow rate. All screened amino acids (**Table 8**) are eluting at the same time whereas a typical run time is 1.5–2 min per sample. Every analyzed amino acid

The isotopic-labeled standards are closely related to the structure of the analyzed amino acids and have similar physicochemical properties to the target amino acids, but can be distinguished by mass spectrometry as they have a different

is assayed with the corresponding stable isotopic labeled standard.

rable to the reference IEC and thus adaptable to the clinical laboratory.

**124**

**Figure 8.**

*Derivatization of alanine with n-butanol.*

*Amino acids analyzed by FIA-MS/MS for the standard newborn screening panel and their stable isotopic labeled internal standards.*

mass to charge ratio (*m*/*z*) (**Table 8**). They are added at a known quantity, and the response of each analyzed amino acid is normalized by the response of the matching internal standard. This type of normalization reduces a systematic error due to the poor recovery and decreases multiple matrix effects. The inclusion of internal standards also corrects a batch to batch variability due to the sample preparation and overall raises the accuracy and precision of the assay.

The tandem mass spectrometer has five basic components: the ion source where all molecules are a subject to the soft ionization, a mass analyzer that separates analytes based on their mass to charge ratio (Q1), a collision cell where molecular ions encounter an inert gas and undergo fragmentation (Q2), a second mass analyzer to separate fragments produced in the collision cell (Q3), and a detector. In collision cell, most of the screened butylated α-amino acids form a common and a very specific fragment of 102 Da (**Figure 9**). The tandem mass spectrometer then can be set to scan a constant mass difference of 102 Da and to produce a spectrum of the molecular ions derived from those amino acids that lost 102 Da in the collision cell (Q2) (**Figure 9**). Butylated amino acids with a basic side chains such as ornithine, citrulline loose ammonia and butyl formate in the

#### **Figure 9.**

*Schematic presentation of tandem mass spectrometer. Phenylalanine (as butyl ester) looses 106 Da in the collision cell. When mass spectrometer operates in neutral loss scanning mode, it scans Q1 and Q3 in a synchronized manner. The mass difference of 102 Da (corresponds to a neutral fragment common to the most amino acids) passing through Q2 remains constant.*

**Figure 10.** *Sulfur amino acids and their disulfides.*

collision cell (*m*/*z* 119). For glycine and arginine, the most intensive signal corresponds to the loss of 56 and 161 Da, respectively. All these specific losses or transitions can be detected by different and highly specific tandem mass spectrometer's scans in the single analysis.

The main limitation of the FIA-MS/MS is inability to differentiate amino acids that share the same *m*/*z* such as leucine/isoleucine and hydroxyproline (butylated derivatives *m*/*z* 188), alanine/sarcosine (butylated derivatives *m*/*z* 146) and in a more extended profiles glutamine/lysine (butylated derivatives *m*/*z* 186), proline/ asparagine (butylated derivatives *m*/*z* 172). Also, FIA-MS/MS is not applicable for cysteine and homocysteine analysis since these amino acids are not stable and react to form cystine and homocystine (**Figure 10**). During the ionization process, cystine and homocystine produce double charged molecules and it complicates the analysis.

Due to the high sensitivity and selectivity, there are more mass spectrometrybased techniques are available for the amino acids analysis, although because of extensive sample preparation or limited number of amino acids covered, these methods are not widely used in clinical laboratories. Gas chromatography mass spectrometry (GCMS) [36], capillary electrophoresis mass spectrometry (CEMS) [37], ion pairing (IP)-LC-MS/MS, HILIC-LC-mass spectrometry [38] and two column LC-MS/MS methods [39], ion pairing (IP)-LC-HRMS (TOF) [40] can be successfully applied for the physiological amino acids analysis although with some limitations.
