PHYTOCHEMICAL SCREENING AND GC-MS ANALYSIS OF CHEMICAL CONSTITUENTS OF THE METHANOL FRACTION OF ALSTONIA BOONEI AND GLYPHAEA BREVIS LEAVES.

ABSTRACT

Medicinal plants are various plant used in herbals and thought by some to possess medicinal properties due to their bioactive ingredients. This study was aimed at identifying the phytochemical compounds present in the methanol fractions of Alstonia boonei and Glyphaea brevis leaves using GC-MS technique. The phytochemical analysis of the Alstonia boonei fraction revealed the presence of tannins (3.02±0.28 mg/g), saponins (2.26±0.21 mg/g), alkaloids (3.68±0.23 mg/g), flavonoids (4.22±0.18 mg/g), terpenoids (2.20±0.14 mg/g) and glycosides (1.94±0.08 mg/g). Similarly phytochemicals present in the methanol fraction of Glyphaea brevis leaves includes; alkaloids (5.28±0.72 mg/g), saponins (2.07±0.08 mg/g), flavonoids (3.94±0.06 mg/g), terpenoids (2.15±1.10 mg/g) and cardiac glycosides (3.05±0.21 mg/g). Steroids were not detected in both fractions. The GC-MS analysis of Alstonia boonei leaves and Glyphaea brevis leaves revealed nine peaks and twelve peaks respectively which represent the nine and twelve bioactive compounds. The major bioactive components present in the methanol fraction of Alstonia boonei were; 3, 7, 11, 15-Tetramethyl-2-hexadecen-1-ol (9.41 %), n-Hexadecanoic acid (15.69 %), Phytol (8.66 %), 9,12-Octadecadienoic acid (Z,Z)- (26.57 %) and Squalene (32.32 %), while the minor compounds present were; 1-Dodecanol,

3,7,11-trimethyl-1 (1.35 %), Cyclopropane nonanoic acid, 2-[(2-butylcyclopropyl) methyl]-, methyl ester (1.96 %), and Phen-1, 4-diol, 2, 3-dimethyl-5-trifluoromethyl-(1.40 %). The major bioactive compounds identified in the methanol fraction of Glyphaea brevis leaves were Cycloheptasiloxane tetradecamethyl- (5.88 %), Hexadecanoic acid, methyl ester (6.55

%), 9, 12, 15-Octadecatrienoic acid, 2, 3-dihydroxypropyl ester, (Z,Z,Z)- (9.13 %), Phytol (24.57 %) and 9, 12, 15-Octadecatrienoic acid (37.12 %), while the minor compounds present were; Cyclooctasiloxane, hexadecamethyl (1.50 %), Cyclononasiloxane Octadecamethyl- (1.49 %), Ethanol, 2-(9-Octadecenyloxy)-,(Z)- (2.88 %), 9, 12, 15-Octadecatrienoic acid, 2,

3-bis[(trimethylsiliy)oxy] propyl ester, (Z,Z,Z)-(1.86 %), Hexadecanoic acid, 1- (hydroxymethyl)-1, 2-ethanediyl ester (3.51%) and Linoleic ethy ester (3.61 %). Alstonia boonei and Glyphaea brevis leaves are well known for there potential pharmacological properties. Amongst these bioactive properties possessed by these plants include; antimicrobial (3, 7, 11, 15-Tetramethyl-2-hexadecen-1-ol), anti-inflammatory (3, 7, 11, 15- Tetramethyl-2-hexadecen-1-ol), antioxidant (n-Hexadecanoic acid), anticancer (9, 12- Octadecadienoic acid (Z,Z)-)  and antitumour (Squalene). These properties exhibited by these plants are attributed to the presence of inherent bioactive components. Thus, pure compounds or standardized extracts or fractions from these plants possibly can provide unlimited opportuninties for new drug because of the unmatched availability and chemical diversity of bioactive principles.

CHAPTER 1

INTRODUCTION

Medicinal plants are various plant used in herbals and thought by some to possess medicinal properties due to their bioactive ingredients (Edeoga et al., 2005). Bioactive principles are responsible for the therapeutic activities of medicinal plants such as hypoglycaemic, anti- diabetic,  anti-oxidant,  anti-microbial,  anti-inflammatory,  anti-carcinogenic,  anti-malarial, anti-cholinergic, and anti-leprosy activities etc (Negi et al., 2011) Thus, pure compounds or standardized extracts from medicinal plants provide unlimited opportunities for new drug leads because of the unmatched availability and chemical diversity of bioactive principles from the plant kingdom (Ramawat et al., 2009; Sasidharan et al., 2011) Medicinal plants play a great role in human life and have substances that are used for traditional therapeutic and modern drug production purposes in primary health care delivery (Omokhua, 2011). The use of medicinal plants in Nigeria has, significantly increased over recent years as it is easily accessible, cheap and the strong belief that herbal remedies are natural and therefore non- toxic (Ezejiofor et al., 2013). Approximately 20% of known plants have been used in pharmaceutical studies, impacting the healthcare system in positive ways such as treating cancer and harmful diseases (Naczks and Shahidi, 2006). Plants are able to produce a large number of diverse bioactive compounds. High concentrations of phytochemicals, which may protect against diseases, accumulate in fruits and vegetables (Suffredini et al., 2004)

1.1 ALSTONIA BOONEI

Alstonia boonei is a very large, deciduous, tropical-forest tree belonging to the dogbane family,  i.e. Apocynaceae.  It  is  native  to  tropical West  Africa,  with  a  range  extending into Ethiopia and Tanzania.   Its   common   name   in   the   English timber   trade is cheese wood, pattern  wood or stool  wood while  its  common  name  in  the  French  timber  trade is emien (derived from the vernacular of the Ivory Coast). The wood is fine-grained, lending itself to detailed carving; the tree also finds many uses in folk medicine. Like many other members     of     the     Apocynaceae     (a     family     rich     in     toxic     and     medicinal species), A. boonei contains alkaloids and yields latex (Burkhill, 1985).

1.1.1 PLANT DESCRIPTION

Alstonia boonei is a tall forest tree, which can reach 45 metres (148 ft) in height and 3 m (9.8 ft) in girth, the bole being cylindrical and up to 27 m (89 ft) in height with high, narrow, deep-fluted  buttresses.  The  leaves  are  borne  in  whorls  at  the  nodes,  the leaf  shape is

oblanceolate, with the apex rounded to acuminate and the lateral veins prominent and almost at   right   angles   to   the midrib.   The   flowers   are   yellowish-white   and   borne   in   lax terminal cymes.  The  fruits  are  pendulous,  paired,  slender follicles up  to  16  centimetres (6.3 in)  long,  containing  seeds  bearing  a  tuft  of  silky,  brown  floss  at  either  end  to allow dispersal by the wind. The latex is white and abundant.

1.1.2 CHEMICAL COMPOSITION

Chromatography of bark extracts of Alstonia boonei on silica gel plates with the solvent system AcOEt-MeOH-H2O (150 : 26 : 19) produced 6 separate spots with alkaloid reactions and the alkaloids isolated from the plant include echitamine  and echitamidine, voacangine and akuammidine, Nα-formylechitamidine, and Nα-formyl-12-methoxyechitamidine (Oguakwa, 1984). Echitamine , which is also isolated from the bark of Alstonia scholaris, Alstonia cogenesis, and Alstonia neriifolia, has been assigned the nomenclature (3β,16R)-

3,17-Dihydroxy-16-(methoxycarbbonyl)-4-methyl-2-4(14)-cyclo-3, 4-secoakuammilinium. These alkaloids, especially echitamine, possess a battery of pharmacological and autonomic activities (Ojewole, 1983) including anticancer activities (Keawpradub et al., 1997).

Iridoids  isolated  from Alstonia  boonei include  boonein  and  loganin.  Loganin  is  a  key intermediate in the biosynthesis of indole alkaloids. It is a crystal of melting point of 222-

20223°C [α]D

−82.1 (water). It is freely soluble in water, less soluble in 96%, alcohol and

sparingly soluble in absolute alcohol. It is practically insoluble in ether, pet, ligroin, ethyl acetate, and chloroform.

Boonein is     a     C-9     monoterpenoid α-lactone,     isolated     from     the     bark     of A. boonei (Apocynaceae). The structure was established by chemical and spectroscopic methods and by X-ray analysis. It is a possible precursor in the indole alkaloid biogenesis (Marini- Bettolo  et al., 1983).The triterpenoids isolated from Alstonia boonei include lupeol, ursolic acid, and β-amyrin.

1.1.3 PHARMACOLOGICAL ACTIVITIES

1.1.3.1 ANTI-ARTHRITIC ACTIVITY

In another development, lupeol acetate isolated from the petroleum ether fraction of Alstonia boonei root barks was tested for its anti-arthritic effect in CFA-induced arthritic rats (Rajic  et al., 2000). It was administered orally every 48 hrs (66 mg/kg body wt.) from days 32 to 40 after adjuvant and assessed on day 60. Lupeol acetate was able to return the increase in spleen weight and the reduction in serum alkyl phosphatase to nonarthritic control values.

1.1.3.2 ANTI-INFLAMMATORY ACTIVITY

The anti-inflammatory activity of a Ghanaian anti-arthritic herbal preparation was also investigated  (Kweifio-Okai , 1991). The herbal preparation is made of a boiling water extract from    a    powdered    sample    containing Alstonia    boonei root    bark    (90%), Rauvolfia vomitoria root  bark  (5%),  and Elaeis  guineensis nut  without  pericarp  (5%).  The  herbal preparation was tested intraperitoneally for its anti-inflammatory activity by measuring rat hind paw oedema induced by the subplantar injection of carrageenin in the presence or absence of arachidonic acid. Arachidonic acid increased swelling during the early phase of carrageenin oedema. The extract suppressed the late phase of carrageenin oedema and both phases in the presence of arachidonic acid. These preliminary results are consistent with a herbal preparation known to be used in the management of rheumatoid arthritis (Kweifio- Okai, 1991).

1.1.3.3 ANTI-MALARIAL

Odeku, (2008) reported the anti-malarial property of the stem bark of Alstonia boonei, which could be formulated in tablet form. Odugbemi et al. (2007) studied the anti-malarial activities of Alstonia boonei.

1.1.4 ETHNOBOTANICAL USES

The bark of Alstonia tree is one of the effective analgesic (Abbiw, 1990) herbs available in nature. All the parts of the plant are very useful but the thick bark cut from the matured tree is the part that is most commonly used for therapeutic purposes.The bark of the tree is highly effective when it is used in its fresh form; however, the dried one could equally be used. Therapeutically, the bark has been found to possess antirheumatic, anti-inflammatory, analgesic/pain-killing, antimalaria/antipyretic, antidiabetic (mild hypoglycaemic), antihelminthic, antimicrobial and antibiotic properties (Hadi et al., 2001). A decoction could be sweetened with pure honey and be taken up to 4 times daily as an effective painkiller for the following conditions.

Painful menstruation (dysmenorrhoea), when associated with uterine fibroid or ovarian cysts in women; lower abdominal and pelvic congestion associated with gynaecological problems such as pelvic inflammatory diseases; to relieve the painful urethritis common with gonococcus  or  other  microbial  infections  in  men. Alstonia decoction  also  exerts  a  mild antibacterial effect in this case, relieving the aches and pains associated with malaria fever. Alstonia is taken in the form of preparations that exhibits antipyrexia and anti-malaria effects, to combat rheumatic and arthritic pains. The decoction of Alstonia bark could be taken alone as  an  effective  pain-killing  agent.  A  cold  infusion  made  from  the  fresh  or  dried  bark of Alstonia taken  orally two  to  three times  daily exerts  a mild  hypoglycaemic effect  on diabetic patients. The cold infusion is also administered orally for the purpose of expelling round worms and threadworms (Abbiw, 1990).

The fresh bark of Alstonia could be used in preparing herbal tinctures; it is particularly useful as an effective antidote against snake, rat, or scorpion poison. It is also useful in expelling retained products of conception and afterbirth when given to women. Asthma can be treated with a drink prepared from parts of Trema orientalis and decoction of the bark of Alstonia boonei mixed with the roots and bark of cola and fruits of Xylopia parviflora with hard potash(Abbiw, 1990). The bark decoction of Alstonia boonei is used with other preparations in the treatment of fractures or dislocation (Abbiw, 1990), jaundice, and for inducing breast milk. Its latex  is taken as a purgative. The hardened latex  is used for the treatment of yaws. Alstonia boonei  De Wild is regarded as one of few herbs with potential anti-HIV indicators.  In  some  African  countries Alstonia  boonei is  considered  a  sacred  tree  and worshiped in the forest and hence human beings in those countries do not eat its parts.

1.2 GLYPHAE BREVIS

Glyphaea brevis  is an integral part of folkloric medicine in most parts of Africa and South America  is  used  traditionally  to  manage  various  ailments  such  as  fevers,  gonorrhea, dysentery, stomach troubles, lung troubles, parasitic infections, convulsions, constipation, insect control, etc (Noumi and Yomi, 2001). In the past few years, there has been tremendous increase in research on G. brevis which has led to the identification of its anti-infective (Ngumah et al., 2013), antioxidant (Dakam et al., 2008), anticonvulsant (Ogbonnia et al.,

2003), anti-inflammatory (Obiri et al., 2013), antiproliferative (Konan et al., 2014), hepatoprotective effects (Ojelabi et al., 2012), etc. and a subsequent discovery of some lead compounds of therapeutic importance.

1.2.1 PLANT DESCRIPTION

Glyphaea brevis is a tall, often straggling; branchlets sparsely stellate-puberulous, later glabrous shrub. Leaves ovate, obovate, obovate- lanceolate or oblong to elliptic, 5–25 cm long, 1.5–14 cm wide, long-acuminate at the apex, the acumen up to 3 cm long, rounded to subcordate at the base, subentire or doubly-toothed, thin, nearly glabrous or sparsely puberulous on the venation above, and pressed puberulous beneath, often with 2 sorts of

stellate hairs; lateral nerves in 5–7 pairs with the 2 basal reaching over half way up the blade or beyond; stipules lanceolate, approximately 2 mm long, very deciduous; petiole 1.5–3.5 cm long. Peduncles, 0.5–2 cm long; pedicels, 1–3.3 cm long (Fig. 1). Flowers up to 4.5 cm in diameter; sepals green, yellow inside, oblong, 1.5–2 cm long, 0.3–0.5 cm wide, tomentellous outside; petals golden or lemon-yellow, oblanceolate or narrowly oblong, 1–2 cm long, 4–4.2 mm wide; stamens yellow; style 6 mm long; stigma green. Fruit brown, spindle-shaped, 3.5–

7.6 cm long, 1.2–1.6 cm wide, ridged and beaked. Seeds irregularly ellipsoid, 4 mm long, 3 mm wide, wrinkled when dry (Whitehouse et al., 2001).

1.2.2 CHEMICAL COMPOSITION

Research  into  identification  and  characterization  of  biologically  active  compounds  from higher plants with ethnopharmacological benefits in the treatment of various disease conditions is always being carried out. As such, host of phytochemical investigations by different teams have been done on G. brevis . Phytochemical analyses of the aqueous and ethanol extracts of the stem bark revealed the presence of alkaloids, flavonoids, anthraquinones, saponins, glycosides, steroids, phlobatanins and carbohydrates (Oshomoh et al., 2012). Ngumah et al. (2013) also reported that the ethanol extract of the leaf contained saponins and glycosides.

Chemical investigation of the leaf extract of G. brevis lead to the identification of steroidal compounds, triterpenes, and polyols by Mbosso et al.(2013).

1.2.3 PHARMACOLOGICAL ACTIVITY

1.2.3.1 ANTIFUNGAL ACTIVITY

Mbosso et al. (2013) reported on the antifungal potential of the hexane extract of the leaves of G. brevis from in vitro studies conducted. Again, Ngumah et al. (201     3) investigated the sensitivity of selected fungal pathogens implicated in dry rot of postharvest yam to the leaf extract of G. brevis. By employing the cup-plate method, the sensitivity of cold (extracted at room temperature) and hot (extracted at 60 °C) ethanol leaf extract of G. brevis, against Aspergillus niger, Fusarium oxysporum and Penicillium oxalicum was investigated. The cold ethanol   extract   showed   potency  on   all   test   pathogens   with   a  minimum   inhibitory

concentration (MIC) of 2.24 x 10-3  mg ml-1, 2.24 x 10-3  mg ml-1  and 7.08 x 10-3  mg ml-1

against  A. niger , P. oxalicum and F. oxysporum, respectively. The hot ethanol extract of G.

brevis yielded an MIC of 4.0 x 10-4  mg ml-1, 3.16 x 10-3  mg ml-1  and 1.26 x 10-3  mg ml-1

against A. niger , P. oxalicum and F. oxysporum, respectively (Ngumah et al., 2013).

1.2.3.2 ANTIOXIDANT ACTIVITY

A couple of studies have assessed the possible utilization of G. brevis as a source of phenolic antioxidant. Rapid screening for antioxidant activity of the G. brevis extract was done by spotting a methanol solution of the leaf and stem bark extract on silica gel sheet which was developed in chloroform-methanol (9:1 v/v). The developed TLC plate was sprayed with the stable free radical DPPH in methanol. The antioxidant activity of the extract was observed as clear zones against purple background on the developed TLC plates (Dickson et al., 2011).

Using ferric reducing antioxidant power (FRAP), 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and

2, 2‟- azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), the antioxidant activity of

G. brevis was investigated by Dakam et al. (2008)

The extraction processes entailed the use of different solvents to extract G. brevis to identify the most suitable solvent(s) for maximum yield of phenolics. A two- way extraction protocol that employed the usage of ethanol and/or water was realized to give a high yield of phenolic compounds in the extract. There was a positive correlation between phenolic content of G. brevis and observed antioxidant activity measured by the three methods stated (Dakam et al.,

2008).

Both the leaf and stem bark extract exhibited antioxidant activities in total phenol content determination,  total  antioxidant  capacity  and  free  radical  scavenging  activity.  The  bark extract  was  higher in  phenol  contents  with  very good  radical  scavenging activity.  Both extracts however exhibited similar capacities in the total antioxidant assay (Dickson et al.,

2011).

This report on the antioxidant effects of the leaves of G. brevis confirms the observations of Dakam et al. (2008) in which different assay protocols were employed. However, the total antioxidant capacity, free radical scavenging activity and the total phenol content of the stem bark extract were reported by Dickson et al. (2011). These studies give scientific credence to its folkloric use in the management of oxidative stress and related degenerative diseases.

As reported by Ogbonnia et al. (2003) and Dakam et al. (2008), the phytochemical screening of the plant revealed the presence of several therapeutically valued constituents including flavonoids and tannins. Therefore, the extract‟s anti-oxidant activity could be contributed by

the presence of flavonoids and tannins which are known to have powerful antioxidant actions

(Mbosso et al., 2010).

1.2.3.3 ANTIBACTERIAL ACTIVITY

There was a first report on the possible antibacterial activity of the hexane extract of the leaves of G. brevis from an in vitro assay (Brown and Rice-Evans,1998). Work done by Oshomoh and Idu established that the aqueous and ethanol extract of the stem bark of G. brevis show a significant antimicrobial activity against Staphylococcus aureus and Streptococcus mutans at concentration of 3.13 mg ml -1  (Oshomoh and Idu, 2012). This potent anti-microbial activity of the stem bark makes it suitable as potential agent for dental care and cleansing.

In a separate study conducted by Dickson et al. (2011) the leaf extract was realized to show considerable inhibition against some Gram- positive organisms but no inhibition against the Gram-negative organisms tested (Dickson et al., 2011). The stem bark however showed inhibition  against  all  Gram- positive bacteria tested  (i.e.  Bacillus  subtilis  NCTC  10073, Bacillus thurigiensis ATCC 13838, Staphylococcus aureus ATCC 25923 and Enterococcus faecalis  ATCC  29212)  with  the  highest  inhibition  against  B.  subtilis  and  Enterococcus faecalis . The bark extract also showed activity against the Gram-negatives, Proteus vulgaris NCTC 4175 and Escherichia coli NCTC 9002. However, Salmonella typhi NCTC 6017 and Pseudomonas   aeruginosa   ATCC   27853   were   resistant   to   the   bark   extract   at   test concentrations (Dickson et al., 2011).

1.2.3.4 ANTIMALARIAL ACTIVITY

Malaria  is  a  disease  caused  by protistan  parasite  which  in  severe  cases  do  progress  to anaemia, coma or death, especially in children and the elderly (World Malaria Report summary).  Like  all  malaria  parasites,  Plasmodium  berghei  is  transmitted  by  female Anopheles mosquitoes. P. berghei has been an important element in the attempt to learn how to manage and eradicate malaria because of the extreme similarity in life cycle and infectious pattern to human infections (Janse et al., 2006). The study by Anjuwon et al. investigated the possible in vivo antimalarial activity of the methanol extract of G. brevis leaves in P. berghei infected mice (Anjuwon et al., 2015).

From their study, they realized that the n-butanol, residual aqueous portion and ethylacetate fractions of the methanol extract exhibited suppressive anti-plasmodial activity with 76.64%,

73.25% and 72.99% inhibition respectively (Anjuwon et al., 2015).

From this study, it can be concluded that G. brevis possesses significant anti-plasmodial activity and could offer lead molecules for research and development of new antimalarial therapies.

1.2.3.5 ANTICONVULSANT ACTIVITY

A study conducted by Ogbonnia et al. (2003) aimed at providing scientific data to back the traditional use of G. brevis as an anticonvulsant. This ethnopharmacological activity of the leaves of G. brevis was investigated in mice. The strychnine and pentylenetetrazole (PTZ) anticonvulsant evaluations were employed to assess the anti- seizure potential of the extract.

Their study showed that a non-lethal dose of 400 mg kg -1 body weight of the extract offered

60% protection to animals against strychnine-induced convulsion. Doses of 400 and 800 mg kg -1 of the extract offered the same degree of protection to the animals in the PTZ-induced convulsion.  Interestingly,  a  longer  seizure  time  (8.73±0.8)  and  shorter  seizure  time (6.20±1.20)  were  obtained  for  800  mg  kg-1   and  400  mg  kg-1   treated  rats  respectively (Ogbonna et al., 2003).

From this study, it is evident that G. brevis has potential anti-seizure activity which offers scientific credence to its folkloric use. G. brevis therefore serves as a possible source of potential anticonvulsant agent.

1.2.4 ETHNOPHARMACOLOGICAL USES

Glyphaea brevis has been indexed in a lot of ethno-botanical research articles. It is used in folklore medicine for a wide range of conditions and has been well documented in a number of research articles. Aside its ethnopharmacological uses such as; Food (Noumi  and Yomi,

2001) Analgesic, vermifuge, diuretic, abortifacient, ecbolic, hepatitis, dyspepsia, ulcer, gonorrhea (Abbiw D, 1990)  Pulmonary troubles (Ogbonnia et al.,  2003)  Stomach troubles (Abbiw, 1990) Antiemetic, veneral diseases, antidote (stings, bites, etc), insecticide, arachnicides, aphrodisiac, appetizer, laxative, sleeping sickness, chest pains (Mshana et al.,

2000)  Cutaneous,  subcutaneous  parasitic  infections,  genital  stimulant/depressant  (Abbiw,

1990)   Febrifuge, dropsy,  swellings, oedema, gout medicine(Abbiw, 1990). G. brevis is widely employed in a number of Agri-horticulture practices such as ornamental usage. In

partially tended Agri- horticulture and cultivated uses, it is used as hedge and markers as well as fodder. The wood of G. brevis is used as building material and the fiber from the stem employed in farming, forestry, hunting and fishing. The twig is chewed as teeth cleanser and the leaf employed in religious and superstitious ceremonies (Abbiw,1990).

1.3 GAS CHROMATOGRAPHY(GC)

Gas Chromatography (GC), is a type of chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium or an un-reactive gas such as nitrogen, and the stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside glass or metal tubing, called a column. The capillary column contains a stationary phase; a fine solid support coated with a non-volatile liquid. The solid can itself be the stationary phase (Syed and Khushnuma, 2014). The sample is swept through the column by a stream of helium gas. Components in a sample are separated from each other because some take longer to pass through the column than others, the detector for the GC is the Mass Spectrometer (MS). As the sample exits the end of the GC column, it is fragmented by ionization and the fragments are sorted by mass to form a fragmentation pattern. Like the retention time (RT), the fragmentation pattern for a given component of sample is unique and therefore is an identifying characteristic of that component. It is so specific that it is often referred to as the molecular  fingerprint.  Gas  chromatography-mass  spectrometry (GC-MS)  is  an  analytical method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. GC can separate volatile and semi-volatile compounds  with  great  resolution,  but  it  cannot  identify them.  MS  can  provide  detailed structural information on most compounds such that they can be exactly identified, but it cannot readily separate them (Syed and Khushnuma, 2014).

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