Derivatization Method Research Articles

MALDI-TOF mass spectrometry imaging for N-glycans on FFPE tissue sections of mouse NASH liver through Sialic acid Benzylamidation.

Glycans play an important physiological role and are drawing attention as biomarkers that capture pathophysiological changes. Glycans can be detected by mass spectrometry, but recently matrix-assisted laser desorption/ionization- mass spectrometry imaging (MALDI-MSI) has enabled the visualization of glycans distribution on tissues. In this study, focusing on sialylated glycan (sialoglycans), we investigated the amidation reaction used to visualize glycans distribution, and developed a method of sialic acid derivatization by benzylamidation which is more sensitive than conventional amidation. Furthermore, we adapted this method for visualizing glycans in formalin-fixed paraffin-embedded (FFPE) liver tissue from normal mice and non-alcoholic steatohepatitis (NASH) model mice using MALDI-MSI. As a result, an increase in the distribution of glycan N-Acetylneuraminic acid-(NeuAc) ions was observed in the NASH mouse liver, and the change in glycan structure in the NASH model was clarified.

Quantification of Amine- and Alcohol-Containing Metabolites in Saline Samples Using Pre-extraction Benzoyl Chloride Derivatization and Ultrahigh Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC MS/MS).

Dissolved metabolites serve as nutrition, energy, and chemical signals for microbial systems. However, the full scope and magnitude of these processes in marine systems are unknown, largely due to insufficient methods, including poor extraction of small, polar compounds using common solid-phase extraction resins. Here, we utilized pre-extraction derivatization and ultrahigh performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) to detect and quantify targeted dissolved metabolites in seawater and saline culture media. Metabolites were derivatized with benzoyl chloride by their primary and secondary amine and alcohol functionalities and quantified using stable isotope-labeled internal standards (SIL-ISs) produced from 13C6-labeled benzoyl chloride. We optimized derivatization, extraction, and sample preparation for field and culture samples and evaluated matrix-derived biases. We have optimized this quantitative method for 73 common metabolites, of which 50 cannot be quantified without derivatization due to low extraction efficiencies. Of the 73 metabolites, 66 were identified in either culture media or seawater and 45 of those were quantified. This derivatization method is sensitive (detection limits = pM to nM), rapid (∼5 min per sample), and high throughput.

Simultaneous determination of three endogenous chiral thiol compounds in serum from humans at normal and stress states using ultrahigh-performance liquid chromatography coupled to quadrupole-Orbitrap high resolution mass spectrometry

Measurement of chiral thiol compounds such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) in human serum plays an important role in the early diagnosis and warning of cardiovascular disease, neurodegenerative disease, and cancer. We developed a novel chiral mass spectrometry derivatization reagent, (R)-(5-(3-isothiocyanatopyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (NCS-OTPP), with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge for the diastereomeric separation of chiral thiol compounds by ultrahigh-performance liquid chromatography coupled to quadrupole-Orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). A novel method was developed for simultaneous determination of three kinds of chiral thiol compounds based on the NCS-OTPP derivatization method. Three kinds of chiral thiol compounds on a YMC Triart C18 (2.0 × 150 mm, 1.9 μm) column with Rs were 1.56–1.68. The protonated precursor to product ion transitions monitored for GSH was m/z 780.16→747.24/473.18, Cys was m/z 594.20→561.18/473.18, and Hcy was m/z 608.21→575.19/473.18. An excellent linearity for all the analytes with correlation coefficients ≥ 0.9995 and suitable precision with inter-day and intra-day coefficients of variation RSDs was 0.83–4.06% and 0.95–3.11%. Satisfactory accuracy with recoveries between 83.73 and 103.35% was observed. The limit of detection (S/N = 3) was 2.4–7.2 fmol. Furthermore, the method was successfully applied to the simultaneous determination of three kinds of free and total thiol compounds in serum from 10 healthy volunteers at normal and stress states.

Application of GC in the Analysis of Carbohydrates

In the mid-1970s and before, Gas Chromatography was the leading technique for analysing carbohydrates in foods until when HPLC start to dominate. Never the less, GC still offers advantages for many applications of food technologists in carbohydrates. The efficiency of the detection of these molecules is reduced as a result of the high polarity and un-volatility of sugars. To overcome these problems, the sugars can be derivative by removing the active hydrogen such as -OH, consequently increasing volatility and improving detectability. Acylation reaction is the most commonly used method of derivatization.

Quantification of cannabinoids in human hair using a modified derivatization procedure and liquid chromatography-tandem mass spectrometry.

The aim of this work was to develop and validate a liquid chromatography-tandem mass spectrometry method for detecting of the main cannabinoids, cannabinol (CBN) and tetrahydrocannabinol (THC) and the primary metabolite 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH) in hair samples. Extraction of the cannabinoids was carried out by a polymeric strong anion mixed-mode solid-phase extraction cartridge and then employing methanolic HCl followed by 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS) as a derivatization procedure of carboxyl and phenolic groups, respectively, offering enhanced sensitivity for the detection of THC-COOH in hair matrices. Formation of a methyl ester increased its lipophilicity and removed the negative charge on the carboxyl group. Calibration curves were prepared over the range of 0.02-4pg/mg of hair for THC and CBN and 0.2-12pg/mg of hair for THC-COOH. The extraction recovery was between 81% and 105% for all compounds. The limit of detection (LOD) and limit of quantification (LOQ) were 2 and 20pg/mg, respectively, for both CBN and THC and 0.1 and 0.2pg/mg, respectively, for THC-COOH, which met the society of hair testing recommendation. Intra-assay and interassay precision were always lower than 4% and 11%, respectively for these cannabinoids, whereas intra-assay and interassay bias were between +14% and -18% and +15% and -12%, respectively. Twenty-seven hair specimens from cannabis users were investigated. The concentrations of CBN, THC and THC-COOH gave ranges of (0.022-2.562ng/mg), (0.049-0.431ng/mg) and (0.222-4.867pg/mg), respectively. This new method of derivatization improves the LOD to ensure detection of the metabolite.

Development of an On-Tissue Derivatization Method for MALDI Mass Spectrometry Imaging of Bioactive Lipids Containing Phosphate Monoester Using Phos-tag.

Matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) is an emerging label-free method for mapping the distribution of diverse molecular species in tissue sections. Despite recent progress in MALDI-MSI analyses of lipids, it is still difficult to visualize minor bioactive lipids including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). Here, we have developed a novel on-tissue derivatization method using Phos-tag, a zinc complex that specifically binds to a phosphate monoester group. MALDI-MSI with Phos-tag derivatization made it possible to image LPA and S1P in the murine brain. Furthermore, we were able to visualize other low-abundance lipids containing phosphate monoester, such as phosphatidic acid and ceramide-1-phosphate. Compared with conventional MALDI-MS, this derivatization produced LPA images with high spatial accuracy discriminating LPA artificially produced during MALDI-MS analysis. In mice with deficiencies in enzymes that degrade LPA and S1P, we observed marked S1P and/or LPA accumulation in specific regions of the brain. Thus, the present study provides a simple and optimal way to reveal the spatial localization of potent bioactive lipid phosphates such as LPA and S1P in tissues.

Normalization of metabolic data to total thymine content and its application to determination of 2-hydroxyglutarate

Our first objective was to develop an approach useful for reliable normalization of 2-hydroxyglutarate (2-HG) intracellular levels. The second objective was to use our data normalization strategy to verify previously published report on the higher d-2-HG level in tumors of colorectal cancer (CRC) patients than in normal colon fragments. We examined various methods of 2-HG level normalization in cell/tissue extracts (number of cells, mass of tissue, total protein). In order to solve the problems with reliable normalization of the 2-HG levels in colon fragments, we proposed a strategy based on relating the concentrations of 2-HG isomers to total thymine concentrations measured by ultra-performance liquid chromatography (UPLC) with UV detection in acid hydrolysates of the cell/tissue extracts. We used a common method of derivatization with diacetyl-l-tartaric anhydride (DATAN) to separate l- and d-2-HG enantiomers. DATAN-derivatized 2-HG was quantitated by UPLC with tandem mass spectrometry (MS/MS) in the selected reaction monitoring (SRM) mode. We observed a linear dependence of the total amount of thymine released from lymphocytes, HCT 116, K562, and PC-3 by acid hydrolysis on their number of cells. Our results showed a significantly higher level of l- and d-2-HG in cancer-free colon than in tumor.

A Bioassay Using a Pentadecanal Derivative to Measure S1P Lyase Activity.

Sphingosine-1-phosphate (S1P) is a unique lipid ligand binding to S1P receptors to transduce various cell survival or proliferation signals via small G proteins. S1P lyase (S1PL) is the specific enzyme that degrades S1P to phosphoethanolamine and (2E)-hexadecenal and therefore regulates S1P levels. S1PL also degrades dihydrosphingosine-1-phosphate (Sa1P), with a higher affinity to produce hexadecanal. Here, we developed a newly designed assay using a C17-Sa1P substrate that degrades into pentadecanal and phosphoethanolamine. For higher sensitivity in pentadecanal analysis, we developed a quantitative protocol as well as a 5,5-dimethyl cyclohexanedione (5,5-dimethyl CHD) derivatization method. The derivatization conditions were optimized for the reaction time, temperature, and concentrations of the 5,5-dimethyl CHD reagent, acetic acid, and ammonium acetate. The S1PL reaction in the cell lysate after spiking 20 µM of C17-Sa1P for 20 min was linear to the total protein concentrations of 50 µg. The S1PL levels (4 pmol/mg/min) were readily detected in this HPLC with fluorescence detection (λex = 366 nm, λem = 455 nm). The S1PL-catalyzed reaction was linear over 30 min and yielded a Km value of 2.68 μM for C17-Sa1P. This new method was validated to measure the S1PL activity of mouse embryonal carcinoma cell lines of the standard cell (F9-0), S1PL knockdown cells (F9-2), and S1PL-overexpressed cells (F9-4). Furthermore, we treated F9-4 cells with different S1PL inhibitors such as FTY720, 4-deoxypyridoxine (DOP), and the deletion of pyridoxal-5-phosphate (P5P), an essential cofactor for S1PL activity, and observed a significant decrease in pentadecanal relative to the untreated cells. In conclusion, we developed a highly sensitive S1PL assay using a C17-Sa1P substrate for pentadecanal quantification for application in the characterization of S1PL activity in vitro.

Chitosan induces delayed grapevine defense mechanisms and protects grapevine against Botrytis cinerea

In the present study, a commercial chitosan soluble in acid solution and obtained from shrimp shell waste, with a molecular weight of 173 kDa and a degree of acetylation of 17%, named as chitosan (173/17), was investigated. Chitosan is a well-known biopolymer whose antimicrobial properties are highly influenced by the molecular weight, degree of acetylation as well as the preparation and derivatization methods used. Chitosan (173/17) was applied on grapevine leaves before Botrytis cinerea inoculation to verify its effectiveness as a preventive treatment against the fungal infection. The expression of a set of defense marker genes, as well as accumulation of stilbene phytoalexins, was investigated. Thanks to its fungistatic and filmogenic properties, chitosan (173/17) protected grapevine leaves against B. cinerea. Moreover, it induced grapevine defense response: three days after the treatment an induction of the jasmonic acid and ethylene-mediated response, a repression of the salicylic acid-mediated signaling, and a transient accumulation of trans-resveratrol were registered. Our data indicate that chitosan (173/17), when used in preventive application, is able to protect grapevine against B. cinerea infection. The effectiveness of chitosan (173/17) as a natural ecofriendly product for the control of B. cinerea on grapevine was demonstrated.

A simple and fast method for metabolomic analysis by gas liquid chromatography-mass spectrometry.

The metabolomic profile is an essential tool for understanding the physiological processes of biological samples and their changes. In addition, it makes it possible to find new substances with industrial applications or use as drugs. As GC-MS is a very common tool for obtaining the metabolomic profile, a simple and fast method for sample preparation is required. The aim of this research was to develop a direct derivatization method for GC-MS to simplify the sample preparation process and apply it to a wide range of samples for non-targeted metabolomic analysis purposes. One pot combined esterification of carboxylic acids with methanol and silylation of the hydroxyl groups was achieved using a molar excess of chlorotrimethylsilane with respect to methanol in the presence of pyridine. The metabolome profile obtained from different samples, such as bilberry and cherry cuticles, olive leaves, P. aeruginosa and E. coli bacteria, A. niger fungi and human sebum from the ceruminous gland, shows that the procedure allows the identification of a wide variety of metabolites. Aliphatic fatty acids, hydroxyfatty acids, phenolic and other aromatic compounds, fatty alcohols, fatty aldehydes dimethylacetals, hydrocarbons, terpenoids, sterols and carbohydrates were identified at different MSI levels using their mass spectra. The metabolomic profile of different biological samples can be easily obtained by GC-MS using an efficient simultaneous esterification-silylation reaction. The derivatization method can be carried out in a short time in the same injection vial with a small amount of reagents.

MTBSTFA derivatization-LC-MS/MS approach for the quantitative analysis of endogenous nucleotides in human colorectal carcinoma cells

Endogenous ribonucleotides (RNs) and deoxyribonucleotides (dRNs) are important metabolites related to the pathogenesis of many diseases. In light of their physiological and pathological significances, a novel and sensitive pre-column derivatization method with N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was developed to determine RNs and dRNs in human cells using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). A one-step extraction of cells with 85% methanol followed by a simple derivatization reaction within 5 min at room temperature contributed to shortened analysis time. The derivatives of 22 nucleoside mono-, di- and triphosphates were retained on the typical C18 column and eluted by ammonium acetate and acetonitrile in 9 min. Under these optimal conditions, good linearity was achieved in the tested calibration ranges. The lower limit of quantitation (LLOQ) was determined to be 0.1–0.4 μM for the tested RNs and 0.001–0.1 μM for dRNs. In addition, the precision (CV) was <15% and the RSD of stability was lower than 10.4%. Furthermore, this method was applied to quantify the endogenous nucleotides in human colorectal carcinoma cell lines HCT 116 exposed to 10-hydroxycamptothecin. In conclusion, our method has proven to be simple, rapid, sensitive, and reliable. It may be used for specific expanded studies on intracellular pharmacology in vitro.

Determination of monosaccharides in Lycium barbarum fruit polysaccharide by an efficient UHPLC-QTRAP-MS/MS method.

Pre-column derivatisation using 1-phenyl-3-methyl-5-pyrazolone (PMP) is a common method for monosaccharide determination. Herein, a specific ultra-high-performance liquid chromatography quadrupole trap tandem mass spectrometry (UHPLC-QTRAP-MS/MS) method was developed and used for the determination of monosaccharide composition of Lycium barbarum polysaccharide (LBP). Exploration of a new efficient method for monosaccharide determination. In our study, hydrochloric acid was used to hydrolyse the polysaccharide obtained from L. barbarum fruits. Principal component analysis (PCA) and technique for order of preference by similarity to ideal solution (TOPSIS) analysis were used for comprehensive evaluation of the samples. The results showed that LBP was composed of seven monosaccharides: galactose, arabinose, mannose, rhamnose, xylose, ribose, and glucose. The linear relationship of the seven monosaccharides was optimum within a certain concentration range. Quantitative recoveries of the seven monosaccharides from the samples ranged from 94.76% to 102.11%. A rapid quantitative detection method was established, in which the hydrolysis time was reduced from 12h to 2h. By using LBP as one of the indexes to evaluate the quality of L. barbarum, L. barbarum from Zhongning County, Ningxia Province, was identified as having the best quality among the varieties tested. The UHPLC-QTRAP-MS/MS pre-column derivatisation method used in this study was simple, accurate, and sensitive, with good repeatability, and can be used for quality evaluation and origin distinction of L. barbarum and other medicinal plants.

Improvements on the Phenylboronic Acid Derivatization Method in Determination of 3-monochloropropane Fatty Acid Esters in Camellia Oil by N-(n-propyl)ethylenediamine Solid Phase Extraction

Pollution of foods with chloropropanol fatty acid esters is a growing international food safety issue, especially contamination with 3-monochloropropane-1, 2-diol (3-MCPD) esters. Traditional GC/MS method using phenylboronic acid (PBA) as a derivatization reagent has been proven to be high selective. However, the disadvantage of this method is that excessive amounts of phenylboronic acid causes the formation of triphenylboroxin that may lead to instrument fouling and reduces the sensitivity of this 3-MCPD detection method. The objective of this study was to optimize a solid phase extraction method after the phenylboronic acid derivatization to illimate the triphenylboroxin formed form excess PBA treatment, and to provide an instrument-friendlier GC-MS method for the detection of 3-MCPD in camellia oil. Different solid phase extraction methods were compared, and (n-propyl)ethylenediamine (PSA) column was selected for its selective in the absorption of fatty acid of ethylenediamine-N-propyl (PSA). Good linearity for 3-MCPD esters detection was found within the concentration range of 0.2 to 10 mg/kg (r2 = 0.9992). The limits of detection (LOD) and the limits of quantification (LOQ) were 0.05 mg/kg and 0.10 mg/kg, respectively. The average recoveries of 3-MCPD esters spiked at 0.25 mg/kg, 1 mg/kg, and 5 mg/kg in pressed camellia oil ranged from 98.83 to 108.79% with a relative standard deviation less than 10%. When this method was applied to the detection of 3-MCPD esters in commercially available camellia oil samples, high amounts of 3-MCPD were present in refined camellia oil samples: the mean content was 1.33 mg/kg, ranging from < 0.034 to 5.47 mg/kg.

Syntheses and medicinal chemistry of azepinoindolones: a look back to leap forward

Nitrogen-containing heterocyclic scaffolds constitute nearly 75% of small molecules which favorably act as drug candidates. For the past few decades, numerous natural and synthetic indole-based scaffolds have been reported for their diverse pharmacological profiles. In particular, indole-fused azepines, termed azepinoindolones, have come under the radar of medicinal chemists owing to their synthetic and pharmacological importance. A plethora of literature reports has been generated thereof, which calls for the need for the compilation of information to understand their current status in drug discovery. Accumulating reports of evidence suggest that compounds containing this privileged scaffold display their cytotoxic effects via inhibition of kinase, topoisomerase I, mitochondrial malate dehydrogenase (mMDH), and tubulin polymerization and as DNA minor groove binding agents. Herein, we endeavor to present a closer look at the advancements of various synthetic and derivatization methods of azepinoindolone-based compounds. We have further extended our efforts to discuss the pharmacological effects of azepinoindolones in the whole range of medicinal chemistry as anti-Alzheimer, anticancer, anti-inflammatory, antidiabetic, antileishmanial, and antipyranosomal agents and as drug delivery vectors. Our analysis of recent advances reveals that azepinoindolones will continue to serve as potential pharmaceutical modalities in the years to come and their substantial pool of synthetic methods will be ever expanding.

Difference spectrophotometric method for the determination of Fluphenazine hydrochloride in tablets using peroxomonosulfate

The oxidative derivatization method using potassium hydrogenperoxomonosulfate for the indirect spectrophotometric determination of Fluphenazine hydrochloride is presented. Potassium hydrogenperoxomonosulfate is introduced as a derivatizing agent for Fluphenazine hydrochloride, yielding the sulfoxide. This reaction product was successfully used for the spectrophotometric determination of the Fluphenazine hydrochloride. The UV spectroscopic detection of the sulfoxide proved to be a more robust and sensitive method. The elaborated method allowed the determination of Fluphenazine hydrochloride in the concentration range of 0.2-30 µg mL-1. The molar absorptivity at 349 nm is 5.6×103 (dm3cm-1mol-1). The limit of quantification, LOQ (10S) is 0.24 µg/mL. A new spectrophotometric technique was developed and the possibility of quantitative determination of Fluphenazine hydrochloride in tablets 5.0 mg was demonstrated. The present method is precise, accurate and excipients did not interfere. RSD for Fluphenazine Hydrochloride 5.0 mg tablets was 1.37 %.

Characterizing the spatial distribution of dipeptides in rodent tissue using MALDI MS imaging with on-tissue derivatization.

A highly efficient on-tissue derivatization method for the matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) of dipeptides in tissues has been developed via a combination of a home-built laser-assisted chemical transfer (LACT) technique and hexafluoroisopropanol (HFIP).

2018 : Development of HPLC Derivatization Methods and Systems

2018 : Development of HPLC Derivatization Methods and Systems

Photochemical Formation of Methylhydroperoxide in Dissolved Organic Matter Solutions.

Although it is known that the solar irradiation of chromophoric dissolved organic matter (CDOM) solutions generates H2O2, whether or not organic hydroperoxides (ROOHs) are photochemically formed remains unclear. This study employs high-performance liquid chromatography with the postcolumn enzymatic derivatization method to examine whether ROOHs can be formed in CDOM solutions under simulated solar irradiation. Methylhydroperoxide (MHP) is the only identified ROOH under our experimental conditions, and the quantum yields of MHP (ΦMHP) vary from (1.09 ± 0.09) × 10-6 to (4.95 ± 0.11) × 10-6 in the tested CDOM solutions, including four reference natural organic matters and two effluent organic matters. The quantum yields of H2O2 (ΦH2O2) are simultaneously measured, and the ratios of ΦH2O2 to ΦMHP range from 147 to 676. The formation of MHP is highly related to the presence of superoxide radical ions (O2•-) and methyl radicals (CH3•); therefore, a photoformation mechanism of MHP has been proposed. The photochemically generated CH3• reacts with O2 to yield CH3OO•. Subsequently, CH3OO• is reduced to MHP by O2•-. Our results also suggest that the yield of CH3• to MHP under air-saturated conditions is 52% and increases to 98% under oxygen-saturated conditions. The decays of MHP and H2O2 are very similar in terms of photodegradation, hydrolysis, Fenton, and photo-Fenton reactions. This study can be useful to understand the photochemical formation of organic peroxides in surface waters.

Application of Safirinium N-Hydroxysuccinimide Esters to Derivatization of Peptides for High-Resolution Mass Spectrometry, Tandem Mass Spectrometry, and Fluorescent Labeling of Bacterial Cells.

Mass spectrometry methods are commonly used in the identification of peptides and biomarkers. Due to a relatively low abundance of proteins in biological samples, there is a need for the development of novel derivatization methods that would improve MS detection limits. Hence, novel fluorescent N–hydroxysuccinimide esters of dihydro-[1,2,4]triazolo[4,3-a]pyridin-2-ium carboxylates (Safirinium P dyes) have been synthesized. The obtained compounds, which incorporate quaternary ammonium salt moieties, easily react with aliphatic amine groups of peptides, both in solution and on the solid support; thus, they can be applied for derivatization as ionization enhancers. Safirinium tagging experiments with ubiquitin hydrolysate revealed that the sequence coverage level was high (ca. 80%), and intensities of signals were enhanced up to 8-fold, which proves the applicability of the proposed tags in the bottom–up approach. The obtained results confirmed that the novel compounds enable the detection of trace amounts of peptides, and fixed positive charge within the tags results in high ionization efficiency. Moreover, Safirinium NHS esters have been utilized as imaging agents for fluorescent labeling and the microscopic visualization of living cells such as E. coli Top10 bacterial strain.

Effect of Different Derivatization Protocols on the Calculation of Trophic Position Using Amino Acids Compound-Specific Stable Isotopes

Amino acids compound-specific nitrogen stable isotope analysis (AA-CSIA) is an emerging tool in ecology for understanding trophic system dynamics. While it has been successfully used for several independent studies across a range of environments and study locations, researchers have encountered calculation issues for determining trophic position values. Most studies introduce modifications to the constants of trophic position equation calculations, but then fail to account for the equation variations when comparing across separate research studies. The broad acceptance of this approach is anchored in an underlying presumption that no addition of the exogenous nitrogen atom occurs in the different methods; and therefore, such variations should not affect the outcome. In this paper, we evaluate the use of the EZfaast amino acid derivatization kit (chloroformate) and compare it to the isotopic results of two other derivatization methods. We highlight new considerations for working with AA-CSIA that might account for some of the variations in the results and lead researchers to modify constants in the equation. This study concludes that developing unique constants per derivatization method is required to have more accurate cross-study comparisons of trophic positions.