Toxin analysis using mass spectrometry

C H A P T E R

15 Toxin analysis using mass spectrometry Thomas A. Blake, Suzanne R. Kalb, Rudolph C. Johnson, John R. Barr Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, Atlanta, GA, United States

Introduction Toxins are produced by living organisms to support different biological needs (Wink and van Wyk, 2008). They are found in numerous forms with a broad array of chemical structures and biological activities. Challenges of toxin analysis include the need for ultrahigh sensitivity (Taylor, 1987) and selectivity (Humpage et al., 2010) in complex biological matrices because toxins can be lethal at a concentration of less than 1 mg/kg of human body mass (Llewellyn, 2006). Direct analysis methods, such as mass spectrometry, are required to confirm the presence of a toxin because toxins cannot be cultured and do not contain DNA. Preanalytical procedures have played a large role in improving the application of mass spectrometry. Toxins may require further purification to increase method sensitivity and remove matrix interferences, digestion may be needed to produce smaller toxin fragments that are compatible with instrument performance capabilities, or a preanalytical reaction may be needed with a substrate to mimic natural activity. As an alternative, prescreening of suspected

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toxin-containing samples may be completed by sensitive, but less specific, immunoassays (Humpage et al., 2010) or other fluorescent techniques (van de Riet et al., 2011) with minimal type II errors to reduce operating costs. While mass spectrometers can be found in numerous configurations, the discussion of these instruments will be limited in this chapter to liquid chromatography tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF).

Toxins The chemical structures of toxins are diverse, and there are numerous methods to classify them such as their respective biological sources, modes of toxicity, molecular mass, and structural characteristics (Wink and van Wyk, 2008). For the purposes of this discussion, toxins will be described as small molecule (Hall et al., 1990), peptide (Abbott et al., 2018; Helfer et al., 2014; Jehl et al., 1985; Yilmaz et al., 2014; Thapa et al., 2014; Rodriguez et al., 2015; Hoggard et al., 2017; Himaya and Lewis, 2018; Aili et al.,

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FIGURE 15.1 Representative chemical structures from the three toxin categories. (A) Small molecules (saxitoxin); (B) peptides (a-amanitin); and (C) proteins (ricin A chain [not including glycans]).

2014), or protein toxins (Fig. 15.1). (Schiavo et al., 1993c; Wang et al., 2014, 2015; Kalb et al., 2015a). From a detection perspective, it is important to consider each toxin’s unique chemical structure and stability characteristics when developing a sample preparation and analysis approach. Information on how each toxin interacts with the human body is also useful because it can serve as the basis for measuring toxin activity, as seen with botulinum neurotoxin (BoNT) (Kalb et al., 2015a) and ricin (Kalb et al., 2015a; Wang et al., 2016). Paralytic shellfish poisoning (PSP) toxins are small molecule toxins which include saxitoxin (STX) and other STX congeners (Humpage et al., 2010; Llewellyn, 2006; Hall et al., 1990; Jansson and Astot, 2015; Dell’Aversano et al.,

2004). With an LD50 i.p. (mouse) of 10 mg/kg, STX is one of the most potent PSP toxins (Humpage et al., 2010). Human PSP usually results from consuming STX-contaminated seafood, and exposures may be recognized by the rapid onset of clinical symptoms such as tingling in the lips, gastroenteritis, respiratory paralysis, and possibly death (Humpage et al., 2010; Coleman et al., 2018). STX reversibly inhibits sodium channels in the body and is subsequently excreted intact in urine (Johnson et al., 2009). Sample preparation must be compatible with the high water solubility and alkaline instability of the toxin (Jansson and Astot, 2015). Because STX is part of a group of more than 30 related toxins, the distribution of these toxins has been used as a selective “fingerprint” for

III. Methodology

Introduction

source attribution purposes (Humpage et al., 2010; Hall et al., 1990; Deeds et al., 2008). a-Amanitin, with an LD50 i.p. (mouse) of 100 mg/kg (Wink and van Wyk, 2008), is an example of a potent peptide toxin. A heatstable, bicyclic octapeptide with multiple congeners (i.e., amatoxins), a-amanitin, is produced by species of the genera Amanita, Galerina, and Lepiota (Wink and van Wyk, 2008; Wieland, 1986; Wieland and Faulstich, 1978). Animal and human exposure usually occurs after ingestion of amatoxin-containing mushrooms (accidental or intentional) with the majority of fatal poisonings caused by Amanita phalloides (Wink and van Wyk, 2008; Defendenti et al., 1998; Vo et al., 2017). Symptoms of amatoxin poisoning are unique and include an asymptomatic period during which protein reserves in the body are depleted due to inhibition of RNA polymerase II. After the body’s protein concentration reaches a critical level, severe gastroenteritis, liver failure, and death may follow. a-Amanitin (918 Da) may be detected intact in human urine following a poisoning and can be directly measured by mass spectrometry. a-Amanitin is slightly hydrophobic, heat and pH stable, and can be readily extracted from aqueous matrices. There are nine reported forms of amanitin (Filigenzi et al., 2007), with the a form being one of the most abundant. Because there is a natural distribution of amatoxins, it may be possible to develop this distribution into a toxin fingerprint for attribution purposes. BoNTs are protein toxins (Databank, 2019) with a mass about 500 times larger than STX. The toxic dose for an average adult is estimated to be about 70 mg through oral consumption (Herrero et al., 1967). Human exposure to BoNT results from ingestion of food containing these toxins (Prevention CfDCa, 1998; Schiavo et al., 2000), inhalation of the toxins, or through colonization of a wound (Prevention CfDCa, 1998) or the gastrointestinal tract of infants or immunocompromised individuals by Clostridium botulinum (or other species of BoNTproducing Clostridium). If the bacteria colonize the human body, they continue to generate the

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BoNT toxin, further poisoning the host. Because of the high molecular weight of the toxin, BoNT is primarily excreted through stool. This matrix contains the highest concentrations of BoNT and is commonly used for determining human exposure to BoNT; however, serum is also an important clinical matrix (i.e., specimen) as some forms of botulism are not associated with toxin in the stool. A characteristic symptom of human exposure to BoNT is flaccid paralysis in which the patient is aware of their surroundings. BoNTs are fundamentally different from small molecule toxins because they consist of a heavy chain (100 kDa) and light chain (50 kDa). The light chain functions as a zinc metalloprotease that cleaves and inactivates proteins necessary for acetylcholine release. The heavy chain is responsible for both receptor binding via its C-terminal binding domain (Mahrhold et al., 2006; Dong et al., 2006) and for delivering the catalytic light chain to its target via its N-terminal translocation domain (Simpson, 2004). The light chain selectively cleaves neuronal proteins required for acetylcholine release and, although the light chain is responsible for the toxicity, it requires the heavy chain to produce this toxic activity in vivo. BoNTs are currently classified into seven recognized serotypes (A-G), but only serotypes /A, /B, /E, and /F are known to affect humans. BoNT/A, /C, and /E cleave synaptosomalassociated protein (SNAP-25) (Foran et al., 1996; Binz et al., 1994; Blasi et al., 1993; Schiavo et al., 1993a,b; Williamson et al., 1996), whereas BoNT/B, /D, /F, and /G cleave synaptobrevin2 (VAMP-2) (Schiavo et al., 1992, 1993c, 1994; Yamasaki et al., 1994a,b; Kalb et al., 2012). Only BoNT/C is known to cleave more than one protein as it also cleaves syntaxin (Foran et al., 1996; Schiavo et al., 1992, 1994). Cleavage of any of these proteins, which interact to form the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, results in an inability to form this complex thus stopping nerve impulses. Ricin is another dual chain (i.e., A-B) protein toxin that is produced in the seeds of the

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decorative plant Ricinus communis. It has an LD50 in humans estimated to range from 70 to 70,000 mg/kg depending on the route of exposure (Bradberry et al., 2003). Ricin reacts rapidly in the lungs or gastrointestinal tract causing cell death by inhibiting protein synthesis. The toxin has a mass of 64 kDa, with two equivalent mass chains of 32 kDa (A chain and B chain) joined by a disulfide bond. The A chain is responsible for the toxin’s enzymatic activity, which involves the depurination of a single adenosine that is part of a GAGA tetraloop of the 28S ribosomal RNA (Endo et al., 1987; Amukele et al., 2005). This depurination results in the inability of the 28S ribosomal RNA to bind elongation factor 2, resulting in the inhibition of protein synthesis (Montanaro et al., 1975) and leading to the clinical symptoms associated with ricin poisoning. The B chain is responsible for delivering the A chain to its target via binding to a cell receptor (Simmons et al., 1986). The B chain is heavily glycosylated, and this glycosylation is thought to assist in receptor binding. Both chains are needed for toxin activity in vivo. Because ricin is extremely reactive with human cells, it is not excreted intact in urine like small molecule toxins, but it can be detected in aqueous matrices and blood (Kopferschmitt et al., 1983; Lim et al., 2009). TABLE 15.1

Ricinine is an alternative chemical target which may be measured in lieu of ricin. Ricinine is a toxic alkaloid present in castor seeds. It has a low molecular weight (164 Da) and is present at roughly 0.3%e0.8% of the seed mass (Johnson et al., 2005). Determining the level of ricinine present in a sample does not directly detect ricin but confirms that a sample contains components of the castor seed. Advantageously, ricinine is extremely heat and pH stable, making it a more persistent marker than ricin. Because ricinine is a marker for a toxin, the LD50 is not applicable (see Table 15.1). Basic analytical methods primarily determine the identity of the toxin and may include further information such as the quantity of toxin present in a sample and whether the toxin is still biologically active. Before completing these analyses, it is important to consider the matrices in which a toxin may be present. The toxin may only be stable or present in a sample matrix for a short period of time, and the concentrations that are associated with a lethal or sublethal exposure may be outside of the measurement capabilities of the selected assay. pH and temperature of a sample can also directly impact stability of the toxin. Additionally, because the toxins may be part of a class of toxins (e.g., STX, BoNTs), the presence or lack of other toxins in its respective

Summary of toxin examples and mass spectrometry methods. Related Confirmatory or Activity Molecular measured? weight (Da)a,b figures presumptive test

Toxin Natural source of category toxin

Mechanism of toxicity

Saxitoxin

Small Marine dinoflagellate, molecule cyanobacteria

Naþ channel No inhibitor in cells

299

1

Confirmatory, LC-MS/MS

Alphaamanitin

Peptide

Amanita phalloides mushroom

Inhibits RNA synthesis

No

918

1

Confirmatory, LC-MS/MS

Botulinum Protein toxin A

Clostridium botulinum bacteria

Nerve Synapses

Yes

150,000

1

Confirmatory, MALDI-TOF

Ricin

Protein

Ricinus communis plant seeds

Inhibits RNA synthesis

Yes

64,000

1, 2

Confirmatory, MALDI-TOF

Ricinine

N.A.

R. communis plant seeds

N.A.

No

164

1

Presumptive, LC-MS/MS

Name

a

Monoisotopic mass. These masses are approximate for A-B protein toxins. Ricin contains two equivalent mass chains of 32,000 Da. BoNT/A contains a light chain of 50,000 Da and a heavy chain of 100,000 Da. b

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Introduction

class may be very helpful in determining the source of an exposure. Toxin activity measurements by mass spectrometry are primarily focused on high molecular weight protein toxins, which can be denatured and inactivated by heating or chemical treatment (e.g., bleach, solvent, etc.). Because no change in mass occurs following denaturation, mass spectrometry alone cannot differentiate an inactive from an active protein toxin. Reaction of a toxin with enzymatic activity with its substrate provides the basis for determining toxin activity as it results in alteration of the substrate which can be directly measured in the mass spectrometer. While many protein toxins do have activity that can be measured by mass spectrometry, it is important to note that not all protein toxins are enzymes. Some toxins (e.g., staphylococcal enterotoxins) are super antigens and do not have enzymatic activity. Additionally, small molecule toxins are generally not tested for activity by mass spectrometry because the tertiary structure of the toxin does not undergo denaturation like a protein toxin. It is assumed that if the presence of a small molecule toxin in a sample is confirmed by mass spectrometry, then it is considered toxic.

Sample preparation Sample preparation usually includes a purification step and reconstitution of the toxin in a matrix which is compatible with the selected mass spectrometry technique. Sample purification approaches generally include either chemical or immunoaffinity extraction to reduce sample complexity. Microbiological methods (e.g., culture, PCR) are not useful for the analysis of toxins because toxins are not living organisms (Audi et al., 2005). Because of the potent nature of toxins, their detection is performed by trace analysis techniques following selective sample purification. Ideally, sample preparation utilizes small amounts to conserve the sample. Solid-phase extraction (SPE) is ideally suited for removing low molecular weight toxins from

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a liquid matrix and can be used to filter samples to remove impurities or bind, rinse, and preconcentrate toxins (Telepchak et al., 2004). Common SPE sorbents include C18, C8, and derivatized silica (e.g., cation exchange, anion exchange). The format of SPE has traditionally been in a polypropylene tube with a flow-through design, although 96-well plate formats are widely available for increasing analytical throughput. The SPE cartridges or plates are disposable, low cost, and are frequently used for only one sample. A common sample size is about 0.1e1 mL and may require preextraction centrifugation to remove particulates. Quantitative SPE methods use internal standards which are precisely added to samples before any preparation steps. The fixed ratio of the toxin signal to that of the internal standard during mass analysis compensates for variability introduced by sample manipulation and extraction, as well as variability during the analysis itself. Stable isotopeelabeled internal standards are chemically identical to the target compound, but they can be readily differentiated in a mass spectrometer. The use of stable isotopeecontaining (e.g., 13C, 2 H, 15N) internal standards is collectively referred to as isotope dilution, and such internal standards need to be optimized for sensitivity, accuracy, and cost (Wang et al., 2014). Toxins may also be extracted using immunomagnetic separation (IMS), which offers much greater selectively than SPE (Bragg et al., 2018). IMS consists of using toxin-specific antibodies conjugated to a magnetic particle and a simple magnet (Bjorck and Kronvall, 1984). The antibody-coated magnetic particles are mixed with the sample to facilitate binding of the antigen (i.e., toxin) to the antibody. A magnetic field is then applied to separate the magnetic particles from the matrix, and the particles are washed to remove nonspecific matrix components. Sample volumes generally range from 10 mL to 1 mL, with the sample size limited by availability of analyte matrix. Following IMS, the toxin may be directly detected by mass spectrometry (Bragg et al., 2018). Alternatively, the

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activity of the toxin (e.g., BoNT (Kalb et al., 2014) or ricin (Kalb et al., 2015b)) can be measured by allowing the particle-bound toxin to react with an artificial substrate that mimics the toxin’s natural target in the human body. The presence of reaction products and/or unreacted substrate can then be measured by mass spectrometry to evaluate toxin activity. Qualitative and quantitative mass spectrometric measurements of protein toxins may involve treatment with a protease such as trypsin, which digests the toxins into characteristic peptide fragments. These peptides can be compared to electronic databases of peptides to qualitatively confirm the presence of a toxin. Quantitation requires the use of internal standards, which can be added following the digestion step, and usually include stable isotopeelabeled peptides (Norrgran et al., 2009). Isotopically labeled peptides are generally available from commercial suppliers and are more cost-effective than generating isotopically labeled protein toxins.

Mass spectrometry Mass spectrometry is a highly sensitive analytical technique which can generate both qualitative data related to toxin mass and structure and quantitative data related to the concentration in a sample (Skoog and Leary, 1992). Qualitative analysis by mass spectrometry requires as much or more toxin than quantitative analysis. Stable-isotope internal standards compensate for sample-to-sample variability resulting from autosamplers and chromatographic variation and, as a result, increase analytical sensitivity. The most selective forms of mass spectrometry rely on either tandem (Dell’Aversano et al., 2004; Seto and KanamoriKataoka, 2005) or high-resolution mass analyzer configurations (Skoog and Leary, 1992). Tandem mass spectrometers incorporate multidimensional analysis by performing multiple stages of mass analysis. A widely used example of a tandem mass spectrometer is the triple quadrupole. Tandem mass spectrometry experiments

dramatically increase an analysis method’s selectivity and sensitivity by decreasing interferences. High-resolution instruments (e.g., MALDI-TOF or Orbitrap mass spectrometers) possess the ability to differentiate nominally similar ions that cannot be differentiated by low-resolution mass spectrometers such as triple quadrupole instruments. This resolution likewise dramatically decreases background interferences and increases method selectivity and sensitivity. High-performance liquid chromatography (HPLC or LC) further increases the selectivity of tandem mass spectrometers (Skoog and Leary, 1992). Besides facilitating delivery of the sample to the mass spectrometer, HPLC further concentrates and purifies the toxin before analysis and sequentially delivers toxin fractions to the mass spectrometer. The separation process on the analytical column is critical to the effectiveness of the method and is based on the selective partitioning of compounds between a solid stationary phase and a liquid mobile phase. The stationary phase is typically comprised of derivatized silica particles similar to those used in SPE but of much higher quality, more uniformity, and smaller particle sizes. HPLC also typically operates at much higher pressures than SPE. Because the effluent from the HPLC is a liquid and the mass spectrometer is a vacuumbased instrument, an ion source, which facilitates evaporation of the solvent and ionizes the toxin, is needed before mass analysis. Electrospray ionization (ESI) is the interface commonly used for the trace analysis of toxins; the configuration of the HPLC followed by ESI and tandem mass spectrometry is commonly abbreviated as LC-ESI-MS/MS or more simply LC-MS/MS. Some toxins are more efficiently analyzed following their dissolution onto a solid matrix and direct introduction into the mass spectrometer using MALDI. Key components of MALDI are an organic matrix in which samples are admixed, a laser to ablate the matrix, and a solid support. The matrix is typically an unsaturated carboxylic acid which absorbs laser radiation and is vaporized, causing simultaneous vaporization of the admixed compound. The acidic matrix also

III. Methodology

Specific toxin analysis methods

donates a proton to the target analyte, causing ionization. High-resolution mass analysis is commonly applied with MALDI to compensate for the lower front-end selectively due to the absence of the HPLC separation step. A common configuration for toxin analysis is the combination of MALDI and high-resolution mass analysis using time of flight mass spectrometry (MALDITOF). The selection of an LC-MS/MS or MALDITOF approach is dependent on the requirements of the analytical method. LC-MS/MS is ideally suited for mixtures of compounds that would generate uninterpretable overlapping mass spectra in MALDI-TOF. An example of such a mixture would be a proteomics digestion solution which can contain thousands of peptides. MALDI-TOF analysis is ideally suited for high molecular weight compounds beyond the mass range of a quadrupole instrument (>3000 Da) and up to several hundred 1000 Da. MALDITOF can also be conveniently applied to the analysis of low molecular weight peptides for the sake of convenience or speed, assuming a very clean sample prepared using IMS.

Specific toxin analysis methods Saxitoxin analysis STX can be detected in liquid matrices such as water or human urine using SPE followed by LC-MS/MS analysis (Humpage et al., 2010; Dell’Aversano et al., 2004; Johnson et al., 2009), which is a common approach to the analysis of small molecule toxins. Cation exchange SPE has been shown to be effective for binding STX to carboxylic acids on the silica stationary phase through electrostatic attraction. Binding occurs when both the substrate and target ion are ionized at 2 pH units above the pKa of an acidic stationary phase and 2 pH units below the pKa of the basic toxin. For STX, a pH 6.4 phosphate buffer facilitates efficient toxin extraction. The solvents used during sample preparation, in order of use, are as follows: methanol, then water (to wet the substrate), pH 6.4 buffer (to charge

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the substrate), sample addition, water (to remove excess phosphate and matrix salts), acetonitrile (to remove neutral interferences), and 5% formic acid in methanol (pH ¼ 1 to neutralize the stationary phase and elute the toxin). More recently, hydrophilic interaction liquid chromatography (HILIC) SPE has been shown to be an effective extraction approach for STX (Xu et al., 2018). Following elution of STX from the SPE cartridge with a volatile solvent, nitrogen evaporation using mild heating (45 C) is used to concentrate the toxin and decrease method detection limits. Heavy isotope internal standards of small molecule toxins are difficult to synthesize (Bragg et al., 2015). Instead, microorganisms that produce the toxin can be grown in a heavy isotopee enriched environment (e.g., 15N2) so that the synthesized toxin already has the heavy elements incorporated. In the case of STX, Alexandrium dinoflagellates are grown in a 15N2-enriched medium to generate the 15N7-labeled toxin (van de Riet et al., 2011; Johnson et al., 2009). It is important to note that heavy isotope internal standards need to have a minimum number of labels to avoid cross talk in the mass spectrometer with the unlabeled toxin (Wille et al., 2017). STX can be measured using LC-MS/MS and HILIC (Dell’Aversano et al., 2004) with a high organic mobile phase for optimal retention and resolution of the polar toxin. The limits of detection are low nanograms-per-milliliter (ng/mL) concentrations in urine and water. These concentrations of STX are below the levels expected for significant human toxicity. It should be noted that if a complete fingerprint of all PSP toxins or the analysis of seafood is needed, the SPE scheme must be altered (Dell’Aversano et al., 2004). The fingerprinting of STXs has been previously discussed in detail (Hall et al., 1990) and can be used to differentiate the strain of dinoflagellate, to identify the source of contaminated seafood, and for geographic sourcing. However, the generation of toxins is a transient event, and it may not be possible to definitively identify the organism that produced the toxin if the conditions have changed significantly. An approach

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in this case would be to mimic the conditions in which a particular toxin was generated, using an assumed source organism, and determine if the toxin fingerprint was reproduced.

fingerprint of a sample would be ideal for attribution, there are limited sources for the a and b forms, and a fingerprinting methodology has not yet been reported for LC-MS/MS.

a-Amanitin analysis

Botulinum neurotoxin analysis

a-Amanitin is a hydrophobic bicyclic peptide which is amenable to traditional C18 SPE stationary phase and reversed phase chromatographic separation. A critical problem in the analysis of amatoxins, including a-amanitin, by mass spectrometry is the lack of readily available isotopically labeled forms of the authentic toxins to use as internal standards. This shortcoming is due to a lack of reported pathways for preparing key intermediates for making the bicyclic bridge when synthesizing the peptide. Additionally, sources for growing amatoxin-producing mushrooms in a heavy isotope environment have typically been severely limited. Previous methods have reported simple screening without use of internal standards (Herrmann et al., 2012; Jansson et al., 2012) or quantitation against structurally similar surrogate compounds as internal standards (Helfer et al., 2014; Leite et al., 2013; Tomkova et al., 2015). However, the production of 15 N-labeled a-amanitin has recently been reported in Galerina marginata (Luo et al., 2015). The 15 N-labeled a-amanitin was used in the development of a method for detecting a-, b-, and g-amanitin in human urine (Abbott et al., 2018). Amatoxins are structurally stable compounds, and the mass spectrometry analysis of a-amanitin is challenging due to limited available fragmentation pathways. Both positive (Abbott et al., 2018; Leite et al., 2013; Tomkova et al., 2015) and negative (Helfer et al., 2014) ionization modes have been reported; however, each has advantages and limitations. The reported limits of detection for a-amanitin in urine range from 0.22 to 1 ng/mL (Abbott et al., 2018; Helfer et al., 2014; Defendenti et al., 1998; Leite et al., 2013; Tomkova et al., 2015; Pittman and Johnson, 2010) which is sufficient to detect toxic levels in clinical samples. While measuring the amatoxin

Protein toxins, such as BoNT, can be qualitatively and quantitatively analyzed by detection of peptide sequences that are unique to that protein and by measurement of toxin activity. IMSs are integral to these methods and use serotype-specific antibodies for BoNT to bind these toxins to a ferromagnetic particle. After binding and washing the particles, a tryptic digest of the bound toxin generates peptides which are toxin specific. These peptides are analyzed by LC-MS/MS for confirmation of the mass and the amino acid sequence of each toxin-specific peptide. Quantification requires the use of isotopically labeled peptides as internal standards which are added following the digestion step. Activity measurements of BoNTs are performed in parallel or before tryptic digestion as digestion products are not enzymatically active. For BoNT, activity measurements include incubating the IMS-bound toxin with a peptide substrate corresponding to a shortened version of the toxin’s natural target, either SNAP-25 or VAMP-2 (Kalb et al., 2015a). The peptide substrate is cleaved in a specific location, which is different for each of the BoNT serotypes. The reaction product is then analyzed using MALDI-TOF. Detection of the peptide cleavage products corresponding to specific toxindependent locations indicates the presence of a particular BoNT serotype. As an example, the peptide substrate for BoNT/B is derived from the toxin’s natural target, VAMP-2. It has an amino acid sequence of LSELDDRADALQAGASQFESSAAKLKRKYWWKNLK with a molecular weight of 4025 Da. The singly charged peptide substrate appears at mass/charge (m/z) 4026, and the doubly charged peptide substrate appears at

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Validation of toxin methods

FIGURE 15.2 MALDI-TOF mass spectrum of the BoNT/B peptide substrate in the presence of BoNT/B. The intact substrate is present at m/z 4026 with cleavage products at m/z 1760 and 2284 indicating the presence of BoNT/B.

m/z 2013 (Fig. 15.2). BoNT/B cleaves the peptide substrate between the Q and the F residues. The N-terminal cleavage product, LSELDDRADALQAGASQ, appears at m/z 1760, and the C-terminal cleavage product, FESSAAKLKRKYWWKNLK, appears at m/z 2284. These cleavage products serve as biomarkers to indicate the presence of active BoNT/B in a sample. Additionally, the amount of intact peptide substrate decreases on the formation of the cleavage products.

Ricin analysis Ricin can be qualitatively and quantitatively analyzed using the IMS and tryptic digestion approach described for BoNTs. Ricin activity can be determined by incubating the toxin with an RNA substrate that mimics the toxin’s natural target, 28S ribosomal RNA. The sequence of this substrate (rGrCrGrCrGrArGrArGrCrGrC) has a molecular weight of 4538 Da and forms a stem loop structure with a GAGA tetraloop. When ricin interacts with this GAGA tetraloop, one of the adenosines is depurinated. Depurination results in a mass shift from 4538 to 4420 Da (Kalb et al., 2015b). Detection of the depurinated substrate at m/z 4421 indicates the presence of active ricin in a sample (Fig. 15.3); if the mass

of the substrate remains unchanged, then active ricin is not present. Methods that either directly analyze toxins by detection of specific peptide sequences or by their activity are considered to be confirmatory methods, provided that the enzymatic activity and immunoaffinity capture are specific for that toxin. In contrast, a presumptive or screening method can be valuable from a sensitivity or throughput standpoint. Ricinine is a component of the castor bean and can be monitored to confirm the presence of a caster bean product, but not ricin itself. In contrast to the lengthy analysis of ricin which requires immunoaffinity capture and a tryptic digestion step, the biomarker ricinine can be measured more rapidly (Johnson et al., 2005; Isenberg et al., 2018). Ricinine is also more temperature stable, solvent resistant, and is generally stable at acidic and basic pH conditions. Therefore, it can be detected in matrices, such as urine, in which ricin has been degraded to nonspecific fragments.

Validation of toxin methods The general life cycle of a method includes development, validation, application, updates, and retirement. Some key issues to establish

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FIGURE 15.3 MALDI-TOF mass spectrum of the ricin RNA substrate in the presence of ricin. The unaltered substrate is present at m/z 4539 and is depurinated in the presence of ricin to yield a new peak at m/z 4421.

during method development include identifying the scientific objectives of the method, determining the appropriate toxin biomarker, and obtaining resources (e.g., reagents, proper instruments, personnel) to achieve those goals. Toxin methods are typically trace analysis methods (Taylor, 1987), and the analytical approach must include “fitness of use” or “fit for purpose” considerations (Wille et al., 2017; US Department of Health and Human Services FaDA et al., 2018). Once a method is fully developed, it must be validated, which includes a statistical characterization of all method parameters and stability of the toxin biomarker under expected use conditions. Method validation can only be started once all parameters for a method have been finalized from the method development process. Once validation is completed, a quality assurance program (Wille et al., 2017)

is needed to support the method during application. Updates to the method trigger an appropriate level of revalidation, and methods may be retired if no longer needed (Fig. 15.4). The level of validation for a specific toxin method should be proportional to the impact of its intended purpose. If a method will be used for critical measurements that result in a significant action, such as patient treatment or legal consequences, then validation should be more extensive than a method used for exploratory measurements (Wille et al., 2017; US Department of Health and Human Services FaDA et al., 2018). Screening methods, which are used for identifying samples for further confirmatory testing, should be developed to minimize falsenegative results. Establishing or validating a specific method extends beyond the characterization of quality control materials; scientists

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Validation of toxin methods

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FIGURE 15.4 Method life cycle stages include separate stages of development, validation, and updating. Updating may require another validation stage, and the method may be retired if superseded or no longer needed.

have many perspectives on what constitutes complete method validation and minimum criteria (Wille et al., 2017; Jarman et al., 2018). The most common element of method development is identifying which biomarkers are best for the intended purpose. Biomarkers vary widely and directly impact the interpretation of testing results. Examples of specific biomarkers may be the toxin of interest or a processed part of the toxin. Highly valuable safety data can be obtained by measuring reaction products of the toxin with natural targets. Less specific data, such as biomarkers from the source and/or method of preparation, can also provide valuable information for investigations (Isenberg et al., 2018; Pittman et al., 2012; Fredriksson et al., 2018; Wunschel et al., 2012). Validation often includes establishing the selectivity of a toxin biomarker and stability in a matrix such as urine, blood, feces, extracted food, or environmental samples. Expected storage conditions of samples before and after preparation, as well as freeze-thaw stability experiments, also need to be evaluated (Scientific

Working Group for Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology, 2013; Chan et al., 2004; Budowle and Members, 2003). This can be especially critical if there is a long analysis period, where a toxin preparation may not be stable in an instrument autosampler. There are several documents related to method validation, and these are usually presented as minimum criteria (Wille et al., 2017; Scientific Working Group for Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology, 2013). A strong trend in the last 5 years has been the increased consensus of what is acceptable for method validation (Wille et al., 2017; US Department of Health and Human Services FaDA et al., 2018; Scientific Working Group for Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology, 2013). When considering toxin analysis data and whether it is valid and “fit for purpose,” some key questions to consider may include the following: (1) Were the positive and negative

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quality control materials measured within specified limits? If the quality control materials failed, then none of the reported results are valid; (2) Was the toxin measured in a previously evaluated matrix? If a new matrix is being evaluated, then the toxin stability, extraction recovery, and method accuracy are not known; (3) Are there similar methods available in the peer-reviewed literature? Peer-review is critical to establishing that the method uses accepted scientific principles; and (4) Were the analysts qualified to complete the method? These records are commonly retained for external auditing purposes if a laboratory is accredited.

Current limitations to toxin analysis Instrument manufacturers continue to expand the applications of mass spectrometry for toxin analysis. Instruments are faster and more sensitive and can now measure lower concentrations more precisely than ever before. As a result, mass spectrometry analysis is primarily limited by the availability of samples and appropriate reference standards/materials rather than by instrument performance. More sensitive methodologies are needed to detect toxins for a longer period of time after generation or exposure, especially when toxins are reduced in concentration due to environmental influences, matrix stability, or metabolism. However, because detection methods are statistically evaluated procedures, fundamental challenges will continue to be related to achieving optimal method quality, which will be based on recent recommendations for bioanalytical and forensic testing (Humpage et al., 2010).

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III. Methodology