J. Sep. Sci.

2014, 37, 1411–1418 1411

Mei Wang1 Research Article

Emily J. Carrell1
Zulfiqar Ali1
Bharathi Avula1
Cristina Avonto1
Jon F. Parcher1
Ikhlas A. Khan1,2,3
1 National Center for Natural
Products Research, University
of Mississippi, University, MS,
USA
2 Department of Pharmacognosy,
School of Pharmacy, University
of Mississippi, University, MS,
USA
3 Department of Pharmacognosy,
College of Pharmacy, King Saud
University, Riyadh, Saudi
Arabia
Received December 28, 2013
Revised February 17, 2014
Accepted March 16, 2014
Comparison of three chromatographic
techniques for the detection of mitragynine
and other indole and oxindole alkaloids in
Mitragyna speciosa (kratom) plants
Leaves of the Southeast Asian plant Mitragyna speciosa are used to suppress pain and mitigate
opioid withdrawal syndromes. The potential threat of abuse and ready availability of this
uncontrolled psychoactive plant have led to the need for improved analytical techniques for
the detection of the major active components, mitragynine and 7-hydroxymitragynine. Three
independent chromatographic methods coupled to two detection systems, GC with MS,
supercritical fluid chromatography with diode array detection, and HPLC with MS and diode
array detection, were compared for the analysis of mitragynine and other indole and oxindole
alkaloids in M. speciosa plants. The indole alkaloids included two sets of diastereoisomers: (i)
paynantheine and 3-isopaynantheine and (ii) mitragynine, speciogynine, and speciociliatine.
Two oxindole alkaloid diastereoisomers, corynoxine and corynoxine B, were also studied.
The HPLC and supercritical fluid chromatography methods successfully resolved the major
components with slightly different elution orders. The GC method was less satisfactory
because it was unable to resolve mitragynine and speciociliatine. This separation was difficult
by GC with a liquid stationary phase because these diastereoisomers differ only in the
orientation of an interior hydrogen atom. The observed lack of resolution of the indole
alkaloid diastereoisomers coupled with the likeness of the mass and tandem mass spectra,
calls into question proposed GC methods for the analysis of mitragynine based on solely
GC with MS separation and identification.

Keywords: Chromatography and mass spectrometry / Indole alkaloids / Mitragyna

speciosa / Mitragynine / Oxindole alkaloids
DOI 10.1002/jssc.201301389

1 Introduction
Kratom (Ketum) is a psychoactive natural product prepared
and consumed in various forms from the plant Mitrag-
yna speciosa Korth. The plant is endemic to Malaysia and
Thailand and is used in its native countries to enhance tol-
erance for heat and hard labor [1–3]. Kratom can act as a
psychostimulant or produce antinocicptive effects depending
upon the dosage and form of ingestion [4]. In Japan, “in-
cense” sticks containing kratom have recently appeared in
the drug market [5]. It is commonly used as a readily avail-
able (especially over the internet), economic substitute for
opioids. On the other hand, it is also frequently used to miti-
gate opium withdrawal symptoms and reduce dependence on
other drugs, although various studies have shown that kratom
Correspondence: Dr. Ikhlas A. Khan, National Center for Natural
Products Research, University of Mississippi, MS 38677, USA
E-mail: ikhan@olemiss.edu
Abbreviations: ALS, automatic liquid sampler; APCI, atmo-
spheric pressure chemical ionization; CID, collision-induced
dissociation; DAD, diode array detection; SFC, supercriti-
cal fluid chromatography; UHPLC, ultra high pressure liquid
chromatography
itself is also addictive [6, 7]. The major psychotropic compo-
nents of kratom are the corynantheidine-type alkaloids [8, 9]
mitragynine (6) and its derivative 7-hydroxymitragynine (5).
Other alkaloids found in M. speciosa include paynantheine (3),
speciogynine (7), and speciociliatine (8). The structures and
stereochemistry of some of the indole and oxindole alkaloids
commonly found in kratom are given in Fig. 1.
Numerous techniques have been proposed for the anal-
ysis of the indole alkaloids, especially mitragynine, in
M. speciosa commercial products, plasmas, sera, or urine for
metabolic studies. Chromatographic methods are the most
common, with both HPLC and GC methods having been
published. To date, HPLC has proven to be the most popu-
lar chromatographic method for the analysis of M. speciosa
alkaloids.
Diode arrays have been used [2, 10–12] for the detection
of the principal components of kratom. The techniques are
relatively fast and simple. However, they do not provide suffi-
cient specificity, due to lack of clear definition, and the lower
LOQs were relatively high, i.e. in the range 50–500 ng/mL.
Mass-specific detection with quadrupole [5], linear ion
trap [1, 13, 14], and triple quadruple [15–18] mass spectrome-
ters have all been used for the detection of indole alkaloids in
kratom and products purported to contain kratom.


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1412 M. Wang et al.
Figure 1. Structures and stereochemistry of mirtagynine and
other indole and oxindole alkaloids commonly found in kratom.
Kikura-Hanajiri et al. [5] used LC–MS with a quadrupole
mass spectrometer (in scan mode) to analyze for mitragynine,
7-hydroxymitragynine, and other indole alkaloids in dried
leaves and resin of kratom. To date, this is the only report
of the direct quantitation of mitragynine in raw plant mate-
rial or commercial products of kratom. The chromatographic
method involved the use of gradient elution with methanol
and water containing ammonium formate (pH 3.5) with a
total analysis time of 46 min. Linear ion trap MS has been
used [1, 13, 14] to determine mitragynine and paynantheine
in urine using HPLC for metabolic studies. Ion-trap tech-
nology allowed the use of MSn for quantitation and detailed
structural analysis of the metabolites in 20–30 min.
Lu et al. [15] were the first to use triple quadrupole MS for
the analysis of mitragynine in biological samples. The chro-
matography involved isocratic elution with a methanol/water
mixture (80:20) and a C18 column in approximately 30 min
with an LOQ of 100 ng/mL. More recently, mitragynine

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J. Sep. Sci. 2014, 37, 1411–1418
[16, 17] and 7-hydroxymitragynine [18] were analyzed with
a triple quadrupole mass spectrometer in pharmacokinetic
studies involving the analysis of alkaloids in rat plasma. The
LOQ in these investigations were 10 [18], 1 [16], and 0.2
ng/mL [17]. The chromatographic methods involved isocratic
elution with very high percentage (85–90%) of organic elu-
ent with hydrophilic interaction chromatography columns in
approximately 3 min. LC was used as a sample introduction
mechanism rather than a separation scheme. In such cases,
quantitative analysis was accomplished by means of the sen-
sitivity and selectivity of very expensive and complex MS/MS
detection systems.
The earliest GC method for the analysis of mitragynine
was published in 2005 [19]. This work was from a forensic
laboratory in Malaysia where kratom has proven to be a severe
drug problem. A standard (HP-5) capillary column was used
at relatively high temperature, viz., 200⬚C, programmed to
300⬚C. No quantitation or validation was attempted. However,
the mitragynine eluted as a symmetrical peak in <17 min
despite the polar nature of the indole alkaloid. Mitragynine
was identified by comparison of experimental mass spectra
with library spectra.
Philipp et al. [20] developed a GC–MS method for the
analysis of kratom and/or Krypton in urine. The indole alka-
loid metabolites in urine were trimethylsilylated to increase
the volatility of the parent alkaloids. After metabolic and en-
zymatic processing, up to three sites on an alkaloid could be
silylated. O-Desmethyltramadol, the other component along
with mitragynine in Krypton, was determined in the same
manner.
More recently, an extensive discussion of “smart drugs”
described a GC–MS method for the analysis of mitragynine
in kratom [21]. GC–MS analysis of underivatized mitragy-
nine and other indole alkaloids requires high temperatures
200–300⬚C, so there is little control available for optimization
of resolution of the analytes.
The problems encountered in the HPLC and GC analysis
of indole alkaloids, such as mitragynine, led to the inves-
tigation of supercritical fluid chromatography (SFC) as an
alternative to the two more conventional chromatographic
techniques. Early work with SFC for the analysis of alkaloids
involved only the analysis of indole alkaloids in Madagascar
periwinkle [22], opium alkaloids in poppy straw extracts [23],
and glucobrassicin and its derivatives in mustard flowers [24].
SFC is commonly used with carbon dioxide as a liquid elu-
ent with its solubilizing power enhanced by a modifier such
as methanol. The advantages of SFC include the low viscos-
ity and high diffusivity of CO2 compared to common HPLC
solvents. This allows the use of very small particle columns
without extraordinary pumping pressures. The separations
observed with SFC are often orthogonal with those observed
with HPLC. In general, SFC is faster, more economical, and
less environmentally harmful than HPLC. In the area of nat-
ural products research, it is common for isolation chemists
to use normal phase column chromatography with silica
gel. The isolated fractions, however, are often subsequently
analyzed with reverse phase liquid chromatography
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J. Sep. Sci. 2014, 37, 1411–1418
techniques. The use of SFC in the normal phase mode facili-
tates the comparison of the same chromatographic methods
for isolation and analyses of the extract fractions.
The objective of the current investigation was to provide
an impartial comparison of three chromatographic methods
with two detection systems (DAD and MS) for the analysis
of mitragynine, 7-hydroxymitragynine, and other M. speciosa
indole and oxindole alkaloids in natural products.
2 Materials and methods
2.1 Chemicals and standards
Seven oxindole and indole alkaloids, corynoxine B (1),
corynoxine (2), paynantheine (3), 3-isopaynantheine (4), mi-
tragynine (6), speciogynine (7), and speciociliatine (8), were
isolated from the leaves of Mitragyna speciosa (voucher no.
12433, authenticated by Dr. Vijayshankar Raman) at the Na-
tional Center for Natural Products Research (University of
Mississippi) [25]. The identity and purity of the isolated stan-
dards was established by IR and NMR spectroscopy, LC–MS,
GC–MS, and SFC. The alkaloid 7-hydroxymitragynine (5) was
purchased from Chromadex (Santa Ana, CA). The eluents
were all HPLC grade purchased from Fisher Scientific (Fair
Lawn, NJ).
A solution of all eight alkaloids was prepared and ana-
lyzed with each of the instruments. The identity of the com-
ponent(s) corresponding to each chromatographic peak was
determined by (i) the retention times of pure standards, (ii)
UV spectra, and (iii) EI and atmospheric pressure chemical
ionization (APCI) (+) mass spectra. In many cases, however,
the spectroscopic data were inadequate to differentiate ana-
lytes, especially diastereoisomers, so retention times of pure
standards were recorded where necessary.
2.2 UHPLC–MS–DAD instrumentation
The ultra high pressure liquid chromatography (UHPLC)
system consisted of an Agilent 1290 Infinity series UHPLC
with a diode array detector, binary pump, vacuum degasser,
automatic liquid sampler (ALS), and thermostatted column
compartment (Agilent Technologies, Santa Clara, Califor-
nia). The LC instrument was coupled to an Agilent 6120
quadrupole mass spectrometer with a dual APCI and ESI
interface. The column was an Agilent ZORBAX RRHD SB-
C8 (2.1 × 100 mm, 1.8 ␮m, part no. 820120-006). The column
temperature was 30⬚C. The flow rate was 0.3 mL/min. The
eluent was 20% acetonitrile and water (10 mM ammonium
acetate, pH 7.7) programmed to 60% acetonitrile in 30 min,
and then 100% in 35 min. The APCI (+) scanned mass range
was 100–800 Da. The fragmentor was 120 V, and the capillary
voltage was 4000 V. The drying gas flow rate was 10.0 L/min,
the nebulizer pressure was set to 30 psi, and the drying gas
temperature was 300⬚C. The DAD was set to monitor 220 and
254 nm.

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Liquid Chromatography 1413
2.3 SFC–DAD instrumentation
The SFC chromatographic system was an Agilent 1260 Series
SFC/LC consisting of (i) an Aurora SFC pump with a back
pressure regulator, (ii) a quaternary LC pump, (iii) a binary
LC pump for control of the CO2 and modifier flow rates, (iv)
vacuum degasser, (v) dual thermostatted column compart-
ments with a column selection system for eight columns, (vi)
an ALS with a fixed loop injector, and (vii) a DAD with a 10
mm 13 ␮L cell. The instrument was controlled by Open-
LAB CDS (Rev. C.01.03) software. An Agilent Rx-Sil col-
umn (2.1 × 50 mm, 1.8 ␮m) was used at a backpressure
of 180 bar, 0.5 mL/min flow rate, and 25⬚C. The eluent was
CO2 with methanol containing 10 mM ammonium acetate.
The eluent was programmed from 6 to 15% methanol in
10 min. The injection volume was 2 ␮L from a 5 ␮L fixed
loop.
2.4 GC–MS instrumentation
The gas chromatographic system consisted of an Agilent 7890
GC, ALS, and 5957C Inert XL MSD. The GC column was an
Agilent DB-5MS (5% phenyl methyl polysiloxane; 30 m ×
0.25 mm × 0.25 ␮m). The flow rate was 1 mL/min. The
injection volume was 1 ␮L. The split ratio was 25:1. The
column was programmed from 220⬚C (2 min) to 300⬚C at
8⬚C/min and then held at 300⬚C for 10 min. The scanned
mass range was 35–550 Da.
2.5 Methanol and alkaloid-enhanced extraction
The leaves of M. speciosa (550 g) were extracted with methanol
(3 L) for 24 h at room temperature four times. The solvent
was removed under reduced pressure to yield a dried extract.
An aliquot was suspended in 5% HCl in water and extracted
with ethyl acetate. The water-soluble part was basified (pH
9–10) with liquid ammonia and extracted with ethyl acetate.
The ethyl acetate soluble part, separated from basic media,
was dried under reduced pressure to get an alkaloid-enriched
fraction.
3 Results and discussion
3.1 UHPLC method
Figure 2A illustrates the DAD (220 nm) chromatograms of
the standard mixture, methanol extract, and the alkaloid-
enhanced methanol extract of M. speciosa plant leaves. The UV
spectra of the mitragynine stereoisomers (6–8) were identical
with maximum absorption at 220 nm. The major alkaloids
were well resolved in 30 min with an eluent of acetonitrile and
water with a basic additive (ammonium acetate pH 7.7). The
elution order differed from that previously observed by Arndt
et al. [26] and Kikura-Hanajiri et al [5]. These authors used an
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1414 M. Wang et al. J. Sep. Sci. 2014, 37, 1411–1418

Figure 2. Chromatograms of M. Speciosa (kratom). HPLC–DAD (220nm): (A-1) standard mixture; (A-2) alkaloid extract; (A-3) methanol
extract. SFC–DAD: (B-1) standard mixture; (B-2) alkaloid extract. GC–EI total ion: (C-1) standard mixture; (C-2) alkaloid extract.


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J. Sep. Sci. 2014, 37, 1411–1418 Liquid Chromatography 1415

acidic additive in the aqueous eluent. UHPLC methods for

the analysis of mitragynine are common, well established,
and accurate. However, they are relatively slow, require an
organic eluent, and not specific due to the similarity of the
UV and mass spectra of the mitragynine diastereoisomers.
In a companion study [27], the ESI mass spectra and
collision-induced dissociation (CID) fragmentation patterns
for the indole alkaloids from kratom were determined. The
mass spectra for the diastereoisomers 6–8 were essentially
identical with ESI parent ions at m/z 399.226 and CID ions at
m/z 110.095, 174.090, 226.142, and 238.142. These daughter
ion masses agreed with those observed previously for just
mitragynine alone with a triple quadrupole instrument [15].
The same isospectral properties were found for paynantheine
and isopaynantheine, as well as corynoxine and corynoxine
B. SFC can enhance the time resolution of diastereoisomer
so that retention time can be used to distinguish compounds
with similar absorbance and mass spectra.

3.2 SFC method

No SFC method has been reported previously for the analysis

of mitragynine in plant material. Figure 2B shows the DAD
(220 nm) chromatograms of the mixture of standards and the
alkaloid-enhanced extract of kratom. The eluent was CO2 with
methanol containing 10 mM ammonium acetate. The elu-
tion order was almost the exact reverse of that obtained in the
UHPLC system. The mitragynine was well resolved from its
diastereoisomers, speciogynine and speciociliatine. The elu-
tion was accomplished in 7 min with at most 15% methanol
in the eluent. Although SFC is an unexplored method for
the analysis of indole and oxindole alkaloids, the technique
is faster, provides better resolution, and gives separations or-
thogonal to HPLC techniques. SFC has also been successfully
applied for the separation of enantiomers and diastereoiso-
mers [28–31].
3.3 GC method
Figure 2C shows the total ion (EI) gas chromatogram of the
standard mixture and an alkaloid-enriched kratom extract.
GC is a viable, readily accessible, but not commonly applied
method for the analysis of indole and oxindole alkaloids in
M. speciosa. In this technique, however, there are two major
issues that have to be addressed. One is the high temperature
required to elute the alkaloids and the second is the inad-
equate resolution of the two diastereoisomers, mitragynine
(6) and speciociliatine (8). The high-temperature requirement
coupled to the upper temperature limit of the polymeric GC
stationary phase imposes a severe restriction on the paramet-
ric adjustment of the resolution of an alkaloid mixture such
as kratom. The incomplete resolution of diastereoisomers is
an even more significant problem for any GC method for the
quantitative analysis of mitragynine. As shown in Fig. 1, the
difference between these isomers is only the orientation of
Figure 3. EI MS of (A) mitragynine (6); (B) speciocilatine (8).
the hydrogen atom at the 3-position [mitragynine (3S) and
speciociliatine (3R)]. The other diastereoisomer, speciogy-
nine (7), differs in the orientation of an ethylene group at
the 20-position [mitragynine (20S), speciociliatine (20S) and
speciogynine (20R)]. Thus, in a gaseous system with a liquid
stationary phase, it is easy to isolate speciogynine but difficult
if not impossible to resolve mitragynine from speciociliatine.
The EI mass spectra of the three common M. speciosa
alkaloids (6–8) are available from most GC–MS libraries. The
spectra are similar; however, it is possible to distinguish mi-
tragynine (6) from speciociliatine (8) by the relative abun-
dance of the molecular ion [m/z 397(398)] and the ion result-
ing from the loss of a methyl group (m/z 383). The spectra
of these two stereoisomers are shown in Fig. 3. The ratio of
397(398):383 is <1 for speciociliatine (8) and >1 for mitrag-
ynine (6). These mass spectral features can be used to dis-
tinguish, but not quantify, the stereoisomers in cases where
complete chromatographic resolution cannot be achieved.
Another way to distinguish these two stereoisomers is
by derivatization to allow chromatographic resolution. The
trimethylsilyl derivatization technique suggested by Philipp
et al. [20] was used to partially derivatize mitragynine (6) and
speciociliatine (8). The corynantheidine-type alkaloids have a
single NH site for derivatization. A total ion chromatogram
of a mixture of free and derivatized alkaloids is shown in
Fig. 4A. The EI mass spectra for the two derivatized alkaloids
were identical. The spectrum is given in Fig. 4B. The mass
spectra are essentially identical and agree with that previously
reported by Philipp et al. [20] for single-site derivatives.
Derivatization allows the GC separation of mitragynine


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1416 M. Wang et al.
Figure 4. EI total ion gas chromatograms and spectra: (A-1) free and derivatized mitragynine (6) and speciocilatine (8); (A-2) free and
derivatized mitragynine (6). D6 and D8 represent the trimethylsilyl derivatives. (B) EI spectra of D6 and D8 (identical).
and speciociliatine; however, the chemical processing step
is time consuming, not necessarily quantitative, and the
trimethylsilyl-derivatized components were found to be air
sensitive and unstable.
Previously proposed GC methods [19, 21] for the analy-
sis of underivatized mitragynine have relied solely on mass-
specific detection for the identification of mitragynine in sam-
ples of M. speciosa. Such identification is questionable because
of the nearly identical EI, ESI, and ESI MS/MS spectra of
the three diastereoisomers, mitragynine, speciogynine, and
speciociliatine. Only subtle differences in the EI spectra allow
some differentiation. Interference of speciociliatine with the
analysis of mitragynine could lead to significant errors, which
would not be detected by either DAD or MS detectors. Such
interference would not be a problem if kratom extracts did not
contain speciociliatine; however, it is clear from Fig. 2A and B
that both UHPLC and SFC analyses indicated that significant
amount of speciociliatine was present in the kratom extract.
Thus, any proposed GC method for the analysis of mitragy-
nine should clearly account for any possible interference by
speciociliatine.
A more specific analytical scheme has been developed
by Philipp et al. [20] wherein the indole alkaloids from
kratom in urine samples were TMS derivatized in 1–3 sites

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J. Sep. Sci. 2014, 37, 1411–1418
after metabolic and enzymatic processing. The mass spectra
of equivalently derivatized speciociliatine and mitragynine
were identical; however, the retention times differed by
over 150 retention index units. Chromatographic resolution
of mitragynine and speciociliatine was achieved as TMS
derivatives in urine samples. However, no derivatization
scheme has been previously applied for the analysis of
alkaloids in plant materials.
GC–MS experiments allow the production of EI spec-
tra for the alkaloids. The mitragynine stereoisomer spectra
have been published; however, to our knowledge, the EI spec-
tra of corynoxine B (1), corynoxine (2), paynantheine (3), 3-
isopaynantheine (4), and 7-hydroxymitragynine (5) have not
appeared in the literature. The EI mass spectra of these com-
pounds are shown in Fig. 5.
4 Concluding remarks
Classical LC with bonded stationary phases and aque-
ous/organic eluents especially when coupled to mass-specific
detection systems provides a viable method for the analysis of
M. speciosa alkaloids in plant material as well as fluids, such
as urine, plasma, or serum, for pharmacokinetic studies. The
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J. Sep. Sci. 2014, 37, 1411–1418
resolution of indole alkaloid diastereoisomers is usually satis-
factory although the elution order varies with eluent pH. The
use of MS/MS increases the sensitivity and selectivity of this
method at the cost of complexity and simplicity. However, the
similarity of EI, ESI, and CID mass spectra make identifica-
tion and differentiation of the corynantheidine-type alkaloid
diastereoisomers very difficult. Pure analytical standards are
often necessary for positive identification from retention data;
however, such standards are often obtained only by laborious
isolation from natural products.
SFC with liquid carbon dioxide modified with an organic
liquid as the eluent is an effective but relatively unexplored
method for the analysis of kratom alkaloids in plants. This
method provides that the advantages of mass-specific detec-
tion and MS/MS are applicable to this technique as well as
conventional HPLC. SFC methods are fast, simple, and re-
quire less organic liquids than HPLC.

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Liquid Chromatography 1417
Figure 5. EI spectra of (A) 7-hydroxy
mitragynine (5); (B) corynoxine B (1) and
corynoxine (2; identical); (C) paynantheine (3)
and 3-isopaynantheine (4; identical).
GC is an appealing technique for such analyses. The in-
dole alkaloids can be eluted underivatized from a capillary col-
umn with high efficiency. GC–MS instrumentation is readily
available in most analytical labs; however, standard capillary
columns may not provide adequate resolution of alkaloid di-
astereoisomers, such as mitragynine and speciociliatine, and
the diastereoisomers are difficult to distinguish from the EI
mass spectra. In addition, the temperature range available
for method optimization is limited. Derivatization may be
necessary to overcome poor chromatographic resolution.
While any of the three chromatographic methods com-
pared in this study may be used for the analysis of mitragynine
and other indole and oxindole alkaloids, the methods differ
in their ability to specifically identify mitragynine in the
presence of its stereoisomers speciogynine (7) and specio-
cialiatine (8). Mitragynine is the primary component for
quantitative studies because of its psychoactivity. However,
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1418 M. Wang et al.
any proposed quantitative method for mitragynine should
recognize the possible interference of other diastereoisomers
with undistinguishable UV, EI-MS, ESI-MS, and ESI-
MS/MS spectra. In this case, chromatographic separation is
imperative and should be clearly established in any proposed
analytical method. Proper care should be considered for the
estimation of mitragynine in pharmacokinetic studies as well
as authentication/adulteration of M. speciosa plant products.
This research is supported in part by “Science Based Authenti-
cation of Dietary Supplements” funded by the Food and Drug Ad-
ministration grant number 5U01FD004246, the United States
Department of Agriculture, Agricultural Research Service, Spe-
cific Cooperative Agreement No. 58-6408-02-1-612. The authors
wish to thank Agilent Technologies for provision of the analytical
instrumentation.
The authors have declared no conflict of interest.
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