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Chemical derivatization for forensic drug analysis by GC- and LC-MS.

TABLE OF CONTENTS

   INTRODUCTION
   Application of Chemical Derivatization in GC and GC-MS
   Application of Chemical Derivatization in LC and LC-MS
   Scope and Relevance of This Review

I. DERIVATIZATION REAGENTS AND REACTIONS

   A. Conventional Derivatization Reagents
   B. Derivatization Reagents to Optimize LC, LC-MS Ionization
     Sources, and MS/MS Performance
   C. Practical Considerations

II. CHEMICAL DERIVATIZATION TO IMPROVE ANALYTE'S RECOVERY, STABILITY,
AND COMPATIBILITY WITH CHROMATOGRAPHIC ENVIRONMENT

III. CHEMICAL DERIVATIZATION TO IMPROVE SEPARATION EFFICIENCY OR
ACHIEVE REQUIRED SEPARATION

   A. Improving Separation Efficiency
   B. Achieving Required Separation--Enantiomeric Determination

IV. CHEMICAL DERIVATIZATION HELPFUL TO DETECTION ENHANCEMENT
OR STRUCTURAL/FUNCTIONAL GROUP CHARACTERIZATION

   A. Detection Enhancement
   B. Structural/Functional Group Characterization

V. APPLICATIONS OF MULTIPLE DERIVATIZATION

   CONCLUDING REMARKS
   REFERENCES
   ABOUT THE AUTHORS


INTRODUCTION

Application of Chemical Derivatization (CD) in Gas Chromatography (GC) and GC-Mass Spectrometry (MS) A series of sample preparation steps are often applied to a test specimen (typically with complex matrix) to prepare the analyte for analysis by the instrumental method of choice. One potential step is the conversion of the analyte to a more suitable form (for analysis) through a well-designed CD route. This CD option, thereby incorporated, may inadvertently increase the analytical cost; it may also complicate data interpretation caused by uncertainty on the completeness of an analyte's conversion process and other interfering factors, such as the introduction of impurities. However, drugs are often derivatized prior to their GC methods of analysis to improve their analytes' (a) volatility and stability (e.g., in the GC injection port); (b) chromatographic property and/or separation efficiency; (c) functional group characterization; and (d) analysis by non-mass spectrometric selective detection methodologies (e.g., electron capture and nitrogen-phosphorus detection) [10]. With MS detection in GC-MS (including GC-MS/MS) methodologies, the CD step can also (a) generate favorable mass shift in mass spectra; (b) modify fragmentation pattern; and (c) facilitate the chemical ionization methodology [10].

Application of CD in Liquid Chromatography (LC) and LC-MS

One commonly cited advantage of the LC and LCMS (including LC-MS/MS) methodologies is that highly polar and/or low volatile analytes can be directly analyzed without the CD step. It was soon recognized, however, that CD can significantly benefit the LC-MS methods for the analysis of certain categories of analytes, e.g., highly polar short-chain acids [60] and steroid hormones [7]. Still at an early stage of development, to what extent CD approaches can benefit the LC-MS methodology is yet to be fully realized; nevertheless, numerous studies have already demonstrated that CD can improve stability, optimize recovery and separation, and enhance the detection of many analytes [12].

Scope and Relevance of This Review

Figure 1 [16] illustrates the approximate ranges (in terms of polarity and relative molecular mass) over which GC-MS and electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) LC-MS can be successfully applied to the analysis of selected compound classes. CD has the potential to favorably alter the ionization properties of analytes. For example, organic acids can be derivatized to reduce their polarity for electron impact (EI) GC-MS analysis or derivatized to increase their polarity, making them more amenable to analysis by positive ESI LC-MS.

Having noted this expanded role played by CD, we wish to widen the scope of an earlier review [27] to include CD's applications in LC-MS. Since scientists from the bioanalytical, pharmaceutical, environmental, and food-safety evaluation communities have been mainly responsible for these advances, most analytes included in their studies are not of particular interest to forensic scientists. Nevertheless, comprehending advances made in these areas would help develop CD approaches for the application of LC-MS in forensic science. With this in mind, most LC-MS application examples cited in this review involve the analysis of drugs of forensic interest.

I. DERIVATIZATION REAGENTS AND REACTIONS

A successful CD reagent is consisted of two moieties: a "reacting" component to react with analytes' functional group(s) and a "goal-oriented" component that would render the derivatization product more amenable to achieving the analytical objective. At the time when the CD technology was first developed, it was important to incorporate a moiety that could effectively enhance analytes' detection by the then-popular ultraviolet, fluorescence, and electron capture detection devices. Since many of these moieties are also effective for GC-MS and LC-MS methodologies, many of these reagents are still being widely used at this date. In addition, new reagents, incorporating moieties specifically effective for various forms of MS methods of detection, have been developed. Table 1 [60] provides a summary of CD reagents and CD reaction categories for LC-MS applications that have been reported in the literature [12,16,27,34,50,60,61].

A. Conventional Derivatization Reagents

Information on a variety of conventional CD reagents is widely available in the literature [1,5,9,10,13,24,33, 36,44,45,49,51,55], Many CD protocols can also be obtained from commercial suppliers providing various CD reagents [51,55], Ideally, a CD reaction is simple, rapid, and stoichiometric, with the reagent's reacting moiety readily reacting to analytes possessing labile protons on heteroatoms, with functional groups such as -COOH, -OH, -NH, and N[H.sub.2]--although high-yield derivatization at carbon sites has also been reported [38].

Three major categories of CD reactions commonly used for drug analysis are: silylation, acylation, and alkylation. The popularity of alkylsilyl derivatization techniques in the analysis of illicit drug by GC is highlighted in a very recent review [35]. Summarized in Table 2 are commonly used CD reagents, with brief descriptions of their main characteristics and illustrations of related CD reactions.

The authors have conducted a search of two categories of GC-MS articles, i.e., those with and without CD, published in journals included in PubMed during the 2006-2015 period. Search data presented in Figure 2 (upper) indicate the percentages of articles involving CD approaches range from 7.70 to 4.99%. Total number of articles increases from approximately 1,400 in 2006 to 2,500 in 2014. The 2015 data were obtained on November 27 (with CD) and November 30 (without CD), 2015.

B. Derivatization Reagents to Optimize LC, LC-MS Ionization Sources, and MS/MS Performance

Unique features related to the application of CD in LC-MS include: (a) liquid mobile phase; (b) ionization sources of LC/MS; and (c) MS/MS mode of operation. While improving analytes' stability and extraction/separation is the common goal for both GC-MS and LC-MS operations, effective CD approaches may vary when the mobile phase is changed from a gas to a liquid gradient. Commenting on potential benefits in applying CD to LC-MS analysis of certain categories of compounds, it has been stated that LC-MS

"is more compatible with the aqueous matrix of biological samples than GC-MS. However, direct LC-MS analysis of short-chain polar carboxylic acids, ketones, and aldehydes without derivatization is still hindered by poor chromatographic performance and low ionization efficiency. Reversed-phase LC columns, as the most commonly used LC columns, are not capable of retaining and separating these hydrophilic metabolites effectively, leading to poor or no signals in the mass detector. ... the instability of reactive carbonyl metabolites, such as acetoacetate and oxaloacetate, in the LC system and the ion source, still prevents reliable detection and measurement.... To overcome these challenges, chemical derivatization offers an alternative solution to enhance chromatographic performance, stability, and detectability of carboxylic acids, aldehydes, and ketones in the LC-MS system ..." [34].

Reagents effective for LC-MS application should produce CD products that will ionize effectively under ESI and APCI sources that are commonly used in LC-MS. For sensitive MS/MS detection, the CD product should also fragment intense product ions, upon collision-induced dissociation (CID).

The authors have also conducted a search of two categories of LC-MS articles, i.e., those with and without CD, published in journals included in PubMed during the 2006-2015 period. Search data presented in Figure 2 (lower) indicate the percentages of articles involving CD approaches range from 2.78 to 4.15%. The total number of articles increases from approximately 1,800 in 2006 to 4,500 in 2014. The cutoff date for 2015 data (with and without derivatization) was December 8, 2015. An earlier report on the prevalence of LC-MS articles published between 1999 and 2009 [60] is shown in Figure 3. Data shown in Figure 2 (lower) and Figure 3 were based on different sets of journals, but the data reported for the period (2006-2009) covered by both figures are compatible in general--i.e., comparing to the data shown in Figure 2 (upper), lower percentages of LC-MS articles included the application of CD approaches.

Reviews on suitable CD reagents for the analysis of various compound categories are also available [15,19,53,54]. Representative examples involving the application of CD approaches to the analysis of drugs of abuse by LC-MS are listed in Figure 4. These examples will be further discussed in later sections.

C. Practical Considerations

Several practical considerations, as listed below, should be considered when selecting a CD reagent and/ or CD reaction.

* Safe and easy formation of the derivative with a readily available and inexpensive reagent;

* High yield of a stable product;

*. Mild reaction conditions preventing undesirable reaction to the analyte; and

* No undesirable by-products that may be harmful to the stationary phase.

Thus, historically important diazomethane for forming methyl ester derivatives from carboxylic acids is no longer popular. The reagent is highly toxic, the reaction is hazardous (may cause an explosion), and the reaction products often include artifacts of unsaturated and keto-acids. Toxicity data of many derivatization reagents have been included in a table in the supplement section of a recent review article [50].

Catalysts, such as HCl, B[F.sub.3], and B[Cl.sub.3] are commonly used with alcohols to form ester derivatives of carboxylic acids. The HCl, used or formed as a by-product when trimethylchlorosilane is used as the trimethylsilyl-(TMS-) derivatization reagent, should be removed prior to the introduction of the derivatization product to a GC or a GC/ MS system. Thus, pyridine and dimethylformamide are commonly used as the solvents because they also act as acid scavengers. Similarly, triethylamine or 5% bicarbonate are used as neutralization agents when trifluoroacetic acid is formed in the trifluoroacetyl derivatization process.

Since the TMS derivatives are susceptible to hydrolysis in the presence of moisture (stability decreases in the order of TMS-ethers > TMS-esters > TMS-amines [9], exposure of the derivatization product to the atmosphere should be limited, especially when the derivatives are not analyzed immediately.

II. CHEMICAL DERIVATIZATION TO IMPROVE ANALYTES' RECOVERY, STABILITY, AND COMPATIBILITY WITH THE CHROMATOGRAPHIC ENVIRONMENT

With an isotopical analog of an analyte serving as the internal standard, it is possible to achieve accurate quantitation without full recovery of the analyte from the test specimen. However, low recovery of the analyte from the sample preparation step often results in lower method sensitivity. For some polar compounds, CD approaches could afford a lipophilic product with the derivative easier to extract and concentration during sample preparation [12]. It has also been implicated that the use of organic solvents and acids in extraction and chromatography caused the degradation of penicillins [52].

Similarly, many analytes could not endure the chromatographic environment and maintain their intact structural features. For example, "ring contraction" would occur when native oxazepam was subjected to the high temperature settings often utilized in GC and GC-MS applications [40,57]. Furthermore, carboxylic acids and amines may form strong hydrogen bonds with silanol groups present in the chromatographic system or residues ofsample components left in the injector or column. These undesired interactions often result in peak loss or peak tailing caused by irreversible or reversible adsorption, respectively [21]. Thus, these hydroxyl (free or part of a carboxylic acid) or amine groups are often converted to inactive species prior to their chromatographic analysis. Chromatograms Figures 5A and 5B [21], obtained using a DB-5 column (5% phenyl polysiloxane phase), show the dramatic differences in their chromatographic characteristics of the six amine and alcoholic amine drugs with and without derivatization. Thus, with the DB-5 column, quantitative determinations or even qualitative identifications of these compounds cannot be achieved without prior derivatization.

The "bringing-about compatibility" (through CD) may be mandatory or simply to improve performance characteristics. There is, however, no clear distinction between these two categories. Using a column with a different stationary phase may render the mandatory requirement an option. As an example, barbiturates can be directly analyzed by GC without the CD step; however, barbiturates in their native forms tend to cause adsorption and result in material loss, column contamination, and peak tailing (Figure 6A). Significantly improvement can be obtained [43] with N,N-dimethylation (Figure 6B) prior to chromatographic analysis. The methylation process has also been utilized [20] to add additional information for confirmatory identification purposes. In another application [31], extracts obtained from urine samples screened positive by RIA were first chromatographed without CD; extracts that show the presence of the targeted barbiturates are then derivatized and chromatographed again. With the conformity of chromatographic parameters to the respective controls, the certainty in confirming the presence of these barbiturates is improved.

In LC and LC-MS, highly polar and nonvolatile analytes can easily be introduced into the mobile phase. However, since highly hydrophilic analytes are poorly retained/resolved by reversed-phase columns commonly used in LC operations, they are likely to co-elute with sample matrices, resulting in signal suppression and/or low signal/noise ratio, especially at the low m/z region. Thus, CDs are performed to increase the analytes' retention time and ion mass. A good example of application in this category involved the use of 2-hydrazinoquinoline as a CD agent for an LC-MS-based metabolomics investigation of diabetic ketoacidosis [34].

III. CHEMICAL DERIVATIZATION TO IMPROVE SEPARATION EFFICIENCY OR ACHIEVE REQUIRED SEPARATION

As highly valued "hyphen" analytical technologies, GC-MS and LC-MS rely on the resolving power of the GC/LC components for the MS component to generate quality spectra. Ideally, the analytes of interest would be well resolved and eluted in a relatively short retention window. CD can impact the separation of analytes in two forms: (a) improving the efficiency of the separation; and (b) realizing the required resolution otherwise not achievable.

A. Improving Separation Efficiency

Under a high-volume production environment, targeted analytes should be eluted and well resolved within an approximately 2-6-min retention window using a reasonably high isothermal GC column temperature. Analytes with retention times less than 2 min will likely be interfered by the solvent, while long retention time reduces the number of samples that can be analyzed with a set period of time. Isothermal operation is convenient and more reproducible; it also causes less baseline drift and minimizes chances of gas leaks that may develop as a result of temperature cycling. Operating at a higher GC oven temperature helps maintain a cleaner chromatographic system. Equipped with improved material for sealing components and advanced software, modern instrumentation is now capable of generating highly reproducible temperature programming; ramping of oven temperature is now widely and effectively used, especially for the analysis of samples with multiple components.

Derivatizations are often performed to help achieve the aforementioned analytical parameters. To bring the analytes' retention times into a more desirable range, drugs of low molecular weights may be converted to esters or amides with acids or alcohols of higher molecular weights, while drugs of higher molecular weights may be converted to esters or amides with improved volatility using fluoro-containing acids or alcohols of lower molecular weights.

Simultaneous analysis of ecgonine methyl ester, benzoylecgonine, and cocaine serves as a good example to illustrate how chromatographic efficiency can be improved through CD. Although these three compounds can be directly chromatographed using a DB-5 column [22], the required chromatographic conditions and the resulting chromatogram (Figure 7A) are significantly improved when a CD step (pentafluoropropionic anhydride) is included in the sample preparation process [39]. The chromatogram shown in Figure 7B was obtained using a dimethyl silicone (HP-19091-6-312) fused-silica capillary column with temperature programming from 100 to 225[degrees]C at 50[degrees]C/min. Chromatograms resulting from the derivatized products are significantly improved.

In an LC-based method for the analysis of hallucinogenic ingredients found in Amanita mushrooms [56], ibotenic acid could not be separated from the intrinsic matrices of the mushroom by dansylation alone. Dansylation product was further converted to ethyl ester for satisfactory separation (Figure 4A). It is interesting to note that the authors adopted LC-MS for qualitative identification of ibotenic acid and another active ingredient, muscimol, but chose UV detection for quantification, because the "sensitivity of GPLC-UV is more stable than that of LC/MS, since it may suffer from phenomena such as ion suppression ..." [56].

B. Achieving Required Separation--Enantiomeric Determination

Enantiomeric separation can be successfully achieved by chiral stationary phases; however, many applications are routinely carried out using CD with chiral reagents. The derivatization may not necessarily add an additional step in the analytical process in cases, where derivatization with nonchiral reagents is applied to improve chromatographic characteristics even when a chiral stationary phase is used. This point is well illustrated by enantiomeric analyses of amphetamine and methamphetamine [30].

As a GC-MS example, Figure 8A [29] shows the chromatograms resulting from the combined use of a chiral derivatizing reagent, N-trifluoroacetyl-7-prolyl chloride (7-TPC), and a chiral column. The four possible isomers resulting from the reaction of d- and l-amphetamine with d- and 7-TPC are completely resolved by the ChirasilVal column. This is important because commercial TPC contains a small amount of d-TPC. The elution order of these four isomers in increasing retention time is N-TFA-d-prolyl-d-amphetamine (Da-d), N-TFA-7-prolyl-l-amphetamine (La-l), N-TFA-d-prolyl-l-amphetamine (La-d), and N-TFA-7-prolyl-d-amphetamine (Da-1). The assignments of these four peaks in a chromatogram were based on relative peak sizes. Since the purity of the TPC reagent and the relative concentrations of d- and 7-amphetamine in control samples are known, the relative intensities of Da-d, La-1, La-d, and Da-l are predictable and their corresponding peaks are assigned accordingly

Contrarily, the four isomers resulting from the reaction of d- and 7-methamphetamine with d- and l-TPC are resolved into three peaks (Figure 8B) only. Based on relative intensities and the known quantity injected, these three peaks, in order of increasing retention time, are N-TFA-d-prolyl-d- methamphetamine (Dm-d), N-TFA-7-prolyl-l-methamphetamine/N-TFA-d-prolyl-l-methamphetamine (Lm-l/Lm-d), and N-TFA-7-prolyl-d-methamphetamine (Dm-l). The inability of the Chirasil-Val column to resolve Lm-l and Lm-d is attributed to the replacement of the active hydrogen atom attached to the nitrogen atom by a methyl group. This replacement reduces the efficiency in forming a transient diastereomeric association complex between the substrate and the chiral phase [42].

Figure 8C is a chromatogram of an authentic amphetamine and methamphetamine mixture obtained from an achiral 25-m SP-2100 column. Since Da-l and La-d and Da-l and La-l are enantiomers to each other and not resolved by the achiral column, only two peaks are observed. By observing the relative intensity of these two peaks, it is concluded that the La-l/Da-d pair elute first. Similar assignments are applied to the methamphetamine peaks.

The contribution due to the small amount of d-TPC can be corrected using the following equations [30]:

[A.sub.a,d] = A ([A.sub.a'],d - D) / (A - D); [A.sub.a,l] = A ([A.sub.a'],l - D) / (A - D)

where [A.sub.a,d] and [A.sub.a,l] are the corrected areas for d-and l-enantiomer respectively; [A.sub.a',d] and [A.sub.a',l] are the apparent areas of d- and l-enantiomer obtained from the chromatograms; A=([A.sub.a',d] + [A.sub.a',l])/2; and D is the impurity (Y) of d-TPC in units of peak area and is given by D =Y/100 x ([A.sub.a',d] + [A.sub.a',l]). Thus, with known concentration (Y) of the d-TPC impurity in the chiral derivatization (l-TPC), the observed peak areas for the d- and 7-enantiomers can be corrected, which is helpful to the determination of the exact enantiomeric compositions of d- and 7-enantiomers in the test sample.

Chiral derivatization approach has also been incorporated into an LC-ESI-MSMS method developed for enantiomeric determinations of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolites in urine [41]. In this study, chiral Marfey's reagent, [N.sup.a]-(5-fluoro2,4-dinitrophenyl)-D-leucinamide (d-FDLA), was added to urine without extraction (Figure 4B). The authors reported satisfactory chromatographic separations for the enantiomers of MDMA and its metabolites 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), HMMA glucuronide, and HMMA sulfide. Using the multiple-reaction monitoring mode, the limits of detection of these analytes were achieved at the 0.01-0.03 [micro]g/mL range.

IV. CHEMICAL DERIVATIZATIONS HELPFUL TO DETECTION ENHANCEMENT OR STRUCTURAL/FUNCTIONAL GROUP CHARACTERIZATION

Chemical derivatizations are commonly used to enhance analyte detection, improve quantitation, and facilitate structural/functional group characterization. Fluorinated anhydrides are extensively used to convert alcohols, phenols, and amines to their fluoroacyl derivatives. In GC and GC-MS, while enhancing analyte volatility through the introduction of fluorine atoms may be desirable in some applications, the high volatility of the resulting derivative may prohibit the use of higher operational temperatures and may not always be desirable for the analysis of low-molecular-weight analytes, such as amphetamine and methamphetamine [17]. Furthermore, the negative inductive effects of the fluorine atoms in the derivatized product were found to render the products more susceptible to hydrolysis in the presence of moisture [14,47].

The introduction of these fluorine atoms, however, greatly enhances detection in cases where electron capture detection is adopted [46,48]. With MS as the detection device, well-designed CD products can generate significantly higher ion intensity and the resulting mass spectra may show distinctive characteristics that are not available from parent compounds. That is to say, CD can be used to enhance detection limits and, in other cases, to help elucidate structural features.

A. Detection Enhancement

The formation of fluoroacyl derivatives from alcohols, phenols, and amines, an approach described early and used to improve the limit of detection in GC applications, has also been applied to negative ion chemical ionization (NICI) in GC/MS applications [18,23]. For example, the NICI method generated a signal that is 200-fold higher than the positive chemical ionization (PCI) counterpart when [[DELTA].sup.9]-tetrahydrocannabinol-11-oic acid was analyzed as its pentafluoropropyl/pentafluoropropionyl derivative [28]. Similarly, the NICI signals for the pentafluorobenzoyl derivative of [[DELTA].sup.9]-tetrahydrocannabinol and the pentafluorobenzoyl and tetrafluorophthaloyl derivatives of amphetamine were found to be 328-, 100-, and 678-fold, respectively, higher than those obtained under PCI conditions [28].

The mass spectra of parent and derivatized amphetamine, as shown in Figure 9, illustrate how CD can help produce a favorable mass spectrum for the analytical process [13]. Improved detection of amphetamine can be achieved through the measurement of higher mass ions obtainable only through derivatization. The spectrum of the parent compound exhibits low intensities of ions at higher mass range. Considering the probability of contributions from interfering compounds, the low mass m/z 44 ion is not suitable for quantitation.

Derivatization of morphine by a conventional CD reagent, dansyl chloride, serves as a good example on how CD can enhance the detection of morphine by LC-MS (Figure 4C). In this report [26], endogenous morphine at 25 pg/mL level was quantitated by APCI LC-triple-quad MS using highly selective-reaction monitoring mode.

B. Structural/Functional Group Characterization

Chemical derivatization can be used to preserve the structural/functional group characteristics, generating MS data that are more amenable to interpretation [2]. For example, to prevent ring contraction that may occur at elevated temperatures, the 3-hydroxy group in oxazepam was derivatized with trimethylsilyl [40] or alkyl [57] group in GC/MS analysis.

Mass shifts in the spectra produced by different derivatizing agents can provide extremely useful information for the identification of an unknown compound. For example, N,O-bis-(trimethylsilyl)-acetamide (BSA) and its deuterated counterpart, [d.sub.9]-BSA, were used to derivatize impurity components isolated from bulk heroin samples; the number of trimethylsilyl (TMS) groups attached to the targeted unknown compound was construed based on the mass shifts observed from mass spectra resulting from replacing BSA with [d.sub.9]-BSA as the derivatizing agent [37]. This "mass shift" information (alone with the presence of other characteristic fragments) facilitated the identification of desoxymorphine-A, monoacetyldesoxymorphine-A, diacetyl-desoxymorphine-A, [O.sup.3],[O.sup.6]-diacetylnormorphine, and [O.sup.6]-acetylnormorphine (Structure 1) as impurities in the heroin sample. The same approach was used to characterize [O.sup.6]- and [O.sup.3]-acetylmorphine [6].

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Similarly, compared to the mass spectrum (Figure 10A) ofthe parent compound, the 28 amu mass shift observed in the mass spectrum of the derivatized pentobarbital (Figure 10B) indicates the replacement of two Hs by two methyl groups [31].

As a third example, compared to parent compounds, TMS derivatives of N-substituted barbiturates are found to generate less olefin radical elimination ([[M-41].sup.+] and [[M-55].sup.-]). Instead, the formation of the [[M-15].sup.+] ion is favored, thus making it easier to recognize the molecular weight of the compound under examination [59].

A CD study [25] on the glucuronic acid conjugate of altrenogest, a drug with abuse potential in the horseracing industry, exemplified certain CD LC-MSMS applications could not possibly be achieved by its GC-MS counterpart. In this application, altrenogest glucuronide was converted to an oxime by derivatization with hydroxyammonium chloride (Figure 4D). The protonated molecule of the oxime (m/z 502) was observed in an extracted ion chromatogram of an MS1 scan. The identification of the derivatized glucurinide was confirmed by collision-induced dissociation, yielding a product ion spectrum with the following ions: M/z 326, corresponding to the altrenogest oxime aglycone; m/z 308, suggesting the loss of [H.sub.2]O from m/z 326 or a loss of glucuronic acid from m/z 502; m/z 284, loss of 42 u from the algycon; m/z 291 and 267, corresponding to loss of OH radicals (17 u) from m/z 308 and 284, respectively. The electrospray response of this derivatized phase II metabolite was found to significantly increase its detection sensitivity (down to at least 13 pg/ mL) and facilitated the identification of this metabolite, using LC-MS/MS in the selected reaction-monitoring mode.

V. APPLICATIONS OF MULTIPLE DERIVATIZATION

When more than one active sites are present in the analyte, it is possible to attach multiple "goal-oriented" moieties to the analyte through sequential/ multiple derivatization steps. For example, In a GC-MS study [58], three amphetamines (amphetamine, methylenedioxyamphetamine, phenylpropanolamine) were first derivatizaed by trifluoroacetyl, pentafluoropropionyl, or heptafluorobutyryl. The resulting products were then added to a second moiety, t-butyldimethylsilyl. The primary objective of this study was to examine which approach would generate the most suitable ions for quantitation purpose, when the analytes' deuterated analogs were used as the internal standards. Data obtained led to the conclusion that many, but not all, products derived from "double derivatization" generated ions of higher quality than those derived from "single derivatization".

Another example of multiple CD approach in GC-MS was studied to provide accurate quantitation for compounds that exist as keto- and enol-forms. Certain analytes, such as oxymorphone, oxycodone, hydromorphone, and hydrocodone, exist as keto- and enol-forms, with compositions of these two forms varied dependent on the matrix acidity and other factors. The conversion of the keto-functional to an oxime, followed by subsequent conventional derivatization approaches (Figure 11), has been well studied. This approach was found effective for simultaneous analysis of these and related compounds [11].

In addition to the altrenogest study [25] cited above, two more studies on steroids involving the application of sequential CD for GC-MS and LC-MS analysis of steroids will be discussed here. For the GC-MS study, anabolic steroids in wastewater were derivatized first with hydroxylamine, followed by silylation with HMDS and TFA (Figure 12) [4]. Three acquisition techniques--the full scan, the multiple ion monitoring, and the MRM methods--were compared [3].

For the LC-MS analysis of anabolic androgenic steroids [8] involving sequential CD, 19-norandrosterone (nandrolone main metabolite) was used as the model compound in this discussion. The hydroxyl group in 19-norandrosterone was first esterified with piconilic acid, followed by converting the keto group to Schiff base using either Girard's reagent T or 2-hydrazino pyridine (Figure 4E). Under positive LC-ESI iron-trap MS full-scan mode, the resulting products showed significantly higher magnitude (5-200-times) of ionization efficiency compared with the respective underivatized AAS.

CONCLUDING REMARKS

Chemical derivatization approaches have long played a crucial role in facilitating the applications of GC-based methodologies, especially in expanding the classes of compounds that can possibly be studied by this category of analytical approach. In addition to converting the targeted analyte into a form that is more compatible with the GC environment, CD can also improve the analytes' recovery and detection limit. With MS as the detector, well-designed approaches can provide additional information helpful to structural elucidation

Parallel approaches have been explored for LC-based methodologies. There have been reports on merits of these approaches that are similar to those observed in the GC-based applications; however, in a much smaller scale. According to a literature search reported in 2011 [60], during the 2001-2009 period, the percentages of derivatization-based LC-MS articles ranged from 3.1 to 4.6% of the total number of articles on LC-MS. The percentages are relatively small, but the actual numbers are still quite significant--considering the total number of LC-MS articles published during this time period. It will be interesting to see whether new CD approaches are explored and incorporated into LC-MS related methodologies in the near future.

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D.-L. Lin (1), S.-M. Wang (2), C.-H. Wu (3) *, B.-G. Chen, R. H. Liu (4)

(1) Department of Forensic Toxicology Institute of Forensic Medicine, Ministry of Justice New Taipei City Taiwan

(2) Department of Forensic Sciences, Central Police University Kuei-Shan Hsiang, Taoyuan City Taiwan

(3) Department of Safety, Health, and Environmental Engineering National Yunlin University of Science and Technology Douliou, Yunlin County Taiwan

(4) Department of Justice Sciences University of Alabama at Birmingham Birmingham, Alabama United States of America

* Corresponding author: Dr. Chih-Hung Wu, Department of Safety, Health, and Environmental Engineering, National Yunlin University of Science and Technology, Douliou, Yunlin County, Taiwan; +886 9 2120 7635 (cell phone); g9710813@gmail.com.

Dong-Liang Lin received B.S. and M.S. degrees from the China Medical University (Taichung, Taiwan) in 1982 and 1984, respectively. In 1995, he also received a Ph.D. degree from the Taipei Medical University (Taipei City, Taiwan). Dr. Lin is currently the head of the Toxicology Division of the Institute of Forensic Medicine, Ministry of Justice (MOJ) of the Republic of China (Taiwan), serving as the chief toxicologist for the Institute. Through a competitive examination system, Dr. Lin entered government service in 1987, working in the laboratory division of the MOJ's Bureau of Investigation. He was transferred to his current position in 2001. Dr. Lin has received forensic toxicology and related training from several US institutions, including the Cook County Medical Examiner's Offi ce (Chicago, IL), the New Jersey State Medical Examiner's Offi ce (Newark, NJ), and the US Fish and Wildlife Service Forensics Laboratory (Ashland, OR). Dr. Lin has been actively working on research projects supported by the (Taiwanese) National Science Council, the Council of Agriculture, and the MOJ. He has published more than 30 articles in peer-reviewed journals. Dr. Lin is a member of the American Academy of Forensic Sciences (AAFS) and the International Association of Forensic Toxicologists (TIAFT). He is also a member of the Taiwan Society of Forensic Medicine and the Taiwan Academy of Forensic Sciences (TAFS).

Sheng-Meng Wang received a B.S. degree in forensic science from Central Police University (Taoyuan, Taiwan) in 1988 and a Ph.D. degree in chemistry from National Tsing Hua University (Hsingchu, Taiwan) in 1997. Dr.

Wang is currently professor of forensic science and director of scientifi c laboratories, Central Police University. Dr. Wang has been a visiting associate professor at the Graduate Program in Forensic Science, University of Alabama at Birmingham (Birmingham, AL), and conducted research at the US Federal Aviation Administration's Civil Aerospace Medical Institute (Oklahoma City, OK). Dr. Wang has been working in various areas of forensic toxicology and his current research activities include: evaluation of various chemical derivatization approaches in the sample preparation process, application of solid-phase microextraction to the analysis of drugs in biological fl uids, and the characterizations of drug depositions in various biological specimens.

Since 1988, Dr. Wang has been serving as a laboratory evaluator for the Drug Testing Laboratory Accreditation Program under the auspices of the (Taiwanese) National Bureau of Controlled Drugs. He has also served as the executive secretary for the Taiwan Academy of Forensic Science since 2006.

Chih-Hung Wu received B.S. and M.S. degrees from the Da-Yeh University (Changhua, Taiwan) in 2002 and 2004, respectively. In 2015, he also received a Ph.D. degree from the National Yunlin University of Science and Technology (Yunlin County, Taiwan). Dr. Wu is currently a postdoctoral fellow in the Department of Safety, Health, and Environmental Engineering, National Yunlin University of Science and Technology.

Dr. Wu's research interests are environmental biotechnology, microbial fuel cell technology, and wastewater treatment. After receiving his master's degree, Chih-Hung worked in the mass spectrometry laboratory, Department of Medical Technology, Fooyin University, as a research assistant from January 2005 to June 2007. He has been the key persone for several derivatization articles published during this time. These articles were published in Journal of Chromatography A, Journal of Analytical Toxicology, Clinica Chimica Acta, and Journal of Food and Drug Analysis.

Bud-Gen Chen received a bachelor's degree in applied chemistry and a master's degree in medical technology, both from Fooyin University (Kaohsiung City, Taiwan). Ms. Chen is currently taking a break from her professional pursuits, raising two children at home.

Following the completion of her undergraduate education, Ms. Chen started working in the mass spectrometry laboratory, Department of Medical Technology, Fooyin University, fi rst as a graduate student, then as a research assistant. She has since become very skillful in various aspects related to this analytical technology and is the key author of several articles, with focuses on derivatization approaches, quantifi cation, and compounds of diagnostic values for diabetic patients. These articles were published in Journal of Mass Spectrometry, Journal of the American Society of Mass Spectrometry, Analytical and Bioanalytical Chemistry, Journal of Analytical Toxicology, Journal of Chromatography A, Analyst, and Journal of Neurochemistry. The article published in Journal of Neurochemistry derived from a joint project with researchers at the Max Planck Institute of Psychiatry (Munich, Germany).

Ray H. Liu received a law degree from Central Police University (then Central Police College, Taipei, Taiwan) and a Ph.D. degree in chemistry from Southern Illinois University (Carbondale, IL) in 1976. He is currently serving as the editor-in-chief of Forensic Science Review and professor emeritus in the Department of Justice Sciences, University of Alabama at Birmingham (Birmingham, AL). Before pursuing his doctoral training in chemistry, Dr. Liu studied forensic science under the guidance of Professor Robert F. Borkenstein at Indiana University (Bloomington, IN) and received internship training in Dr. Doug Lucas's laboratory (Centre of Forensic Sciences in Toronto, Canada). Dr. Liu has worked as an assistant professor at the University of Illinois at Chicago (Chicago, IL), as a chemist at the US Environmental Protection Agency's Central Regional Laboratory (Chicago, IL), and as a center mass spectrometrist at the US Department of Agriculture's Eastern Regional Research Center (Philadelphia, PA) and Southern Regional Research Center (New Orleans, LA). He was a faculty member at the University of Alabama at Birmingham for 20 years and retired in 2004 after serving for more than 10 years as the director of the University's Graduate Program in Forensic Science. Dr. Liu's works have been mainly in the analytical aspects of drugs of abuse (criminalistics and toxicology), with a signifi cant number of publications in each of the following subject matters: enantiomeric analysis, quantitation, correlation of immunoassay and GC-MS test results, specimen source differentiation, and development of analytical methodologies. He has authored (or coauthored) several books and book chapters; more than 120 articles in refereed journals; and approximately 150 presentations in scientifi c meetings. He is qualifi ed by the New York State Department of Health to serve as a laboratory director in forensic toxicology and he has served as a technical director in a US drug-testing laboratory that held major contracts with military, federal, local, and private institutions. Dr. Liu has been an active member of the following professional organizations for approximately 30 years: the American Chemical Society, Sigma Xi - The Scientifi c Research Society, the American Academy of Forensic Sciences (fellow), and the American Society for Mass Spectrometry. He is also a member of the Society of Forensic Toxicologists (SOFT) and the International Association of Forensic Toxicologists (TIAFT). Dr. Liu consults with several governmental and nongovernmental agencies, including serving as a laboratory inspector for the US and the Taiwanese workplace drug-testing laboratory certifi cation programs. He is the editor-in-chief of Forensic Science Review and serves on the editorial boards of the following journals: Journal of Analytical Toxicology, Journal of Food and Drug Analysis (Taipei), Forensic Toxicology (Tokyo), and Forensic Science Journal (Taoyuan, Taiwan).

Table 1. Derivatization for LC-MS application (a)

Functional group    Reacting group of
of target           derivatization         Derivatization reaction
compound            reagent

Alcohol             X-CO-R                 Acylation/ sulfonylation
-OH                 X-R                    Alkylation
                    [N.sub.3]-CO-R or      Carbamate formation (acyl
                      O=C=N-R                azide or isocyanate)
                    HO-CO-R or             Esterification (acid or
                      [R.sub.2]-CO           anhydride)
Vicinal diol        [(OH).sub.2]-B-R       Cyclization

[FORMULA NOT
  REPRODUCIBLE
  IN ASCII]
[alpha]-Hydroxyl    [(OH).sub.2]-B-R       Cyclization
  acid
[FORMULA NOT
  REPRODUCIBLE
  IN ASCII]
Phenol              x-S[O.sub.2]-R         Sulfonation/dansylation
[FORMULA NOT        X-CO-R                 Benzoylation
  REPRODUCIBLE      HO-CO-R or             Esterification (acid
  IN ASCII]           [R.sub.2]-CO           or anhydride)
Thiol               R-S-S-R                Disulfide formation
-SH                 X-R                    S-Alkylation
                    S=C=N-R                Sulfur amide formation
Ketone/aldehyde     N[H.sub.2]-R           Imine formation
>C=O                N[H.sub.2]-NH-R        Hydrazone formation
                    N[H.sub.2]-OH          Oxime formation
                    N[H.sub.2]-R +         Amide reduction
                      NaCNB[H.sub.3]
                    SH-R                   Oxathiolane formation

Carboxylic acid     N[H.sub.2]-R           Amide formation
-COOH               N[H.sub.2]-NH-R        Acylhydrazine formation
                    HO-R or [N.sub.2]-R    Esterification
                    tris(Hydroxymethyl)    Oxazoline formation
                    aminomethane

[alpha]-Keto acid   1,2-Di-aminobenzene    Cyclization
-CO-COOH
Amine (primary      X-CO-R                 Acylation
  and secondary)    X-CO-Ph                Benzoylation
-N[H.sup.2]         X-S[O.sub.2]-R         Sulfoamide formation
>NH                 S=C=N-R                Thiourea formation
                    O=C=N-R                Urea formation
s-cis-Diene         Cookson-type reagent   Diels-Alder adduct
-CH=CH-CH=CH-                              formation

Functional group    Structure of
of target           derivatization product
compound

Alcohol             -O-CO-R
-OH                 -O-R
                    -O-CO-N-R

                    -O-CO-R

Vicinal diol        [FORMULA NOT REPRODUCIBLE
                      IN ASCII]
[FORMULA NOT
  REPRODUCIBLE
  IN ASCII]
[alpha]-Hydroxyl
  acid
[FORMULA NOT
  REPRODUCIBLE
  IN ASCII]
Phenol              Ph-O-S[O.sub.2]-R
[FORMULA NOT        Ph-O-CO-R
  REPRODUCIBLE      Ph-O-CO-R
  IN ASCII]
Thiol               -S-S-R
-SH                 -S-R
                    -S-CS-N-R
Ketone/aldehyde     >C=N-R
>C=O                >C=NH-R
                    >C=N-OH
                    >c=n-r

                    [FORMULA NOT REPRODUCIBLE IN ASCII]

Carboxylic acid     -CO-NH-r
-COOH               -CO-NH-NH-R
                    -coo-r
                    [FORMULA NOT REPRODUCIBLE IN ASCII]

[alpha]-Keto acid   [FORMULA NOT REPRODUCIBLE IN ASCII]
-CO-COOH
Amine (primary      >N-CO-R
  and secondary)    >N-CO-Ph
-N[H.sup.2]         >N-S[O.sub.2]-R
>NH                 >N-CS-N-R
                    >N-CO-N-R
s-cis-Diene         [FORMULA NOT REPRODUCIBLE IN ASCII]
-CH=CH-CH=CH-

(a) Reproduced with permission from Ref. [60].

Table 2. Silylation, acylation, and alkylation derivatizing reagents
and characteristics (a)

Reagent and reaction                       Characteristics (b)

Silylation

N,O-Bis(trimethylsilyl)             * React faster and more
trifluoroacetamide                  completely than BSA
(BSTFA)

[FORMULA NOT REPRODUCIBLE IN        * Combine with 1% or 10% TMCS for
ASCII]                              hindered hydroxyl and other
                                    functionalities

Trimethylchlorosilane (TMCS)        * Commonly used as a catalyst

TMS-CI + H-R [right arrow] TMS-R    * Acidic by-product, HCl
+ HCI

N,O-Bis(trimethylsilyl)acetamide    * Mild reaction conditions
(BSA)
                                    * Forms stable products
[FORMULA NOT REPRODUCIBLE IN
ASCII]                              * Volatile by-product, TMS-
                                    acetamide

N-Methyltrimethylsilyl-             * Volatile by-product, TMS-
trifluoroacetamide                  acetamide
(MSTFA)
                                    * Most suitable for volatile
[FORMULA NOT REPRODUCIBLE IN        trace analyte
ASCII]

Trimethylsilylimidizole (TMSI)      * React with hydroxyl group but
                                    not amine

[FORMULA NOT REPRODUCIBLE IN        * Suitable for hindered hydroxyl
ASCII]                              group

Trimethylsilyldiethylamine (TMS-    * Basic reagent for amino and
DEA)                                carboxylic acids

[FORMULA NOT REPRODUCIBLE IN
ASCII]

Hexamethyldisilazane (HMDS)         * A weak TMS donor

TMS-NH-TMS + H-Y-R [right arrow]
TMS-Y-R + TMS-N[H.sub.2]

N-Methyl-N-(t-butyldimethylsilyl)   * Exceptionally strong yet mild
trifluoroacetamide (MTBSTFA)        reagent

[FORMULA NOT REPRODUCIBLE IN        * Stable product, resisting
ASCII]                              hydrolysis

                                    * Combine with 1% t-
                                    butyldimethylchlorosilane
                                    catalyst for hindered alcohol and
                                    amine

Anhydrides (TFAA, PFPA, HFBA, A     * Form fluoroacyl derivatives to
A, TCAA)C                           greatly increase volatility and
                                    improve detectivity in GC and MS,
[FORMULA NOT REPRODUCIBLE IN        especially negative chemical
ASCII]                              ionization

                                    * Often used with bases, such as
                                    triethylamine

Heptafluorobutyrylimidizole         * Reaction fast and mild, work
(HFBI)                              best for phenol, alcohol, and
                                    amine

[FORMULA NOT REPRODUCIBLE IN        * No acidic by-product
ASCII]

N-Methyl-N-bis                      * React rapidly with primary and
(trifluoroacetamide) (MBTFA)        secondary amine; slowly with
                                    alcohol, phenol, and thiol
[FORMULA NOT REPRODUCIBLE IN
ASCII]                              * Mild reaction; inert and
                                    volatile by-products

Pentafluorobenzoyl chloride         * Highly reactive, forming the
(PFBCI)                             most sensitive ECD derivatives of
                                    amine and phenol
[FORMULA NOT REPRODUCIBLE IN
ASCII]                              * Suitable for sterically
                                    hindered functionalities

                                    * Base often used to remove HCl
                                    produced

4-Carbethoxyhexafluorobutyryl       * Form stable product with
chloride (4-CB)                     secondary amine, e.g.,
                                    methamphetamine

[FORMULA NOT REPRODUCIBLE IN        * Add protic solvent to remove
ASCII]                              excess agent

(S)-(-)-N-(Trifluoroacetyl)-        * Widely used for amine drugs
prolyl chloride (l-TPC)
                                    * Proton at the chiral center in
[FORMULA NOT REPRODUCIBLE IN        [alpha]-position to the carbonyl
ASCII]                              group; control storage and
                                    reaction conditions to avoid
                                    racemization through  ketoenol
                                    tautomerization

Propyl chloroformate                * Fast reaction

[FORMULA NOT REPRODUCIBLE IN        * Derivatives water-soluble,
ASCII]                              allowing aqueous washing to
                                    remove by-products

(-)-[alpha]-Methoxy-[alpha]-        * More effective than l-TPC in
trifluoromethylphenylacetic acid    resolving ephedrines and
(MTPA)                              generating ions designating these
                                    analytes
[FORMULA NOT REPRODUCIBLE IN
ASCII]

Alkylation

DMF-Dialkylacetal (n = 1, 2, 3,     * Most commonly used for carboxyl
or 4)                               groups; also reacts with amine,
                                    phenol, amino acid
[FORMULA NOT REPRODUCIBLE IN
ASCII]                              * n = 1-4 for analyte retention
                                    time control

Trimethylanilinium hydroxide        * Commonly used as a flash
(TMAH)                              alkylation reagent

[FORMULA NOT REPRODUCIBLE IN
ASCII]

Tetrabutylammonium hydroxide        * Especially suitable for low
(TBH)                               molecular weight amines

[FORMULA NOT REPRODUCIBLE IN
ASCII]

BF3/Methanol (n-butanol)            * Most commonly used to form
(n = 1 or 4)                        methyl (butyl) ester with acid

[FORMULA NOT REPRODUCIBLE IN
ASCII]

(a) This is a revised version of a table previously published in Ref.
[27].

(b) Information in this column, mostly taken from the reagent
manfuacturer's catelog [51], is included only for general reference
purposes.

(c) Abbreviation: AA = acetic anhydrides; HFBA = heptafluorobutyric
anhydrides; PFPA = pentafluoropropionic anhydrides; TCAA =
trichloroacetic anhydrides; TFAA = trifluoroacetic anhydrides.
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Publication:Forensic Science Review
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Date:Jan 1, 2016
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