Fast Determination of Fumonisin B1 and B2 in Corn Using a Modified QuEChERS Method and LC–MS–MS

A fast analytical method for the simultaneous determination of fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) in corn using a novel QuEChERS method and LC–MS–MS was developed and validated. Samples were extracted with methanol–water (3:1 v/v) by means of ultrasonic extraction. The extract was purified with a novel modified QuEChERS method. Firstly, FB₁ and FB₂ in the extract were retained with primary secondary amine (PSA). Then, FB₁ and FB₂ were released from PSA with 1.0 % formic acid in methanol. The final eluate was diluted with water, and analyzed by LC–MS–MS on a Waters Acquity BEH C₁₈ column with 0.1 % formic acid in water/methanol as mobile phase with gradient elution. Mean recoveries of 83.5–102.4 % with CVs of 3.6–10.5 % were obtained at fortification levels of 2, 50 and 1,000 μg kg⁻¹. The limit of quantification was 2.0 μg kg⁻¹.

     

Tailored 96-well μElution solid-phase extraction combined with UFLC-MS/MS: a significantly improved approach for determination of free 3-nitrotyrosine in human urine

We developed and validated a simple and fast UFLC-MS/MS method for the accurate determination of 3-nitrotyrosine (3-NT) in human urine as a noninvasive biomarker for oxidative stress. The method, involving tailored 96-well μElution solid-phase extraction (SPE) combined with UFLC-MS/MS, allows 3-NT to be determined in biological samples without the need for hydrolysis, derivatization, evaporation, and two-dimensional LC for the first time. Using ammonium acetate (pH?9, 25 mM) as an elution buffer was found to improve SPE selectivity. Fast chromatographic elution of 3-NT with a total run time of 7 min was achieved on a PFPP column (150 mm?×?2.1 mm, 3 μm). This fine-tuned integrated method delivered significantly improved throughput, specificity, and sensitivity while reducing the matrix effect, solvent usage, and waste disposal. Using this simple and rapid method, two plates of urine samples (n?=?192) can be processed within 24 h. The lower limit of quantification for 3-NT is 10 pg/mL, which represents a notable sensitivity enhancement over reported methods. Less than 6.0 % variations for intraday and interday assay precisions and 97.7–106.3 % for accuracies in terms of recovery were obtained. The applicability and reliability of the method were demonstrated by determining the reference range in human urine for 82 healthy people. Considering the noninvasive and inexpensive nature of urine sampling, this novel method could be used to re-evaluate the role of 3-NT as an oxidative stress biomarker in pre-clinical and clinical studies.     

LC–MS/MS Method for Detection of a Highly Selective KCa3.1 Blocker,TRAM-34, in Rat Plasma

1-[(2-Chlorophenyl)diphenylmethyl]pyrazole (TRAM-34) is a highly selective K_Ca3.1 channel blocker. TRAM-34 was commonly used to study the role of K_Ca3.1 in the pathogenesis of disease in vivo, but there was no validated analytical method. Here, we describe the first validated LC–MS/MS analytical method for TRAM-34. Solid-phase extraction (SPE) was performed to extract TRAM-34 from the rat plasma. Chromatographic separation was achieved on the phenyl column. A triple quadrupole mass spectrometer was operated in positive-mode electrospray ionization. There were two multiple-reaction monitoring (MRM) transitions for TRAM-34: m/z 277.2 → 165.1 (for quantification) and m/z 277.2 → 241.2 (for qualification). Bifonazole was used as an internal standard. The lower limit of quantification (LLOQ) achieved was 1 ng mL^?1 and the run time was 7.5 min. The linear range was from 1 to 1,000 ng mL^?1. The pharmacokinetics profile was acquired for rats following an intraperitoneal injection of TRAM-34, with the following pharmacokinetics parameters found: C _max 17.03 ± 1.34 ng mL^?1; T _max 8.67 ± 3.06 h; and T _1/2 13.45 ± 2.72 h. In addition, a suspected metabolite of TRAM-34 was found using this LC–MS/MS method. Given the results of the detailed validation process and its application to TRAM-34 pharmacokinetics, it is clear that a fast, selective, precise, and reproducible TRAM-34 LC–MS/MS analytical method was successfully established.     

Rapid method for the measurement of circulating thyroid hormones in low volumes of teleost fish plasma by LC-ESI/MS/MS

Thyroid hormones are critical regulators of normal development and physiological functioning in all vertebrates. Radioimmunoassay (RIA) approaches have been the method of choice for measuring circulating levels of thyroid hormones in vertebrates. While sensitive, RIA-based approaches only allow for a single analyte measurement per assay, can lack concordance across platforms and laboratories, and can be prone to analytical interferences especially when used with fish plasma. Ongoing advances in liquid chromatography tandem mass spectrometry (LC/MS/MS) have led to substantial decreases in detection limits for thyroid hormones and other biomolecules in complex matrices, including human plasma. Despite these advances, current analytical approaches do not allow for the measurement of native thyroid hormone in teleost fish plasma by mass spectrometry and continue to rely on immunoassay. In this study, we developed a new method that allows for the rapid extraction and simultaneous measurement of total T4 (TT4) and total T3 (TT3) in low volumes (50 μL) of fish plasma by LC/MS/MS. Methods were optimized initially in plasma from rainbow trout (Oncorhynchus mykiss) and applied to plasma from other teleost fishes, including fathead minnows (Pimephales promelas), mummichogs (Fundulus heteroclitus), sockeye salmon (Oncorhynchus nerka), and coho salmon (Oncorhynchus kisutch). Validation of method performance with T4- and T3-spiked rainbow trout plasma at 2 and 4 ng/mL produced mean recoveries ranging from 82 to 95 % and 97 to 105 %, respectively. Recovery of ¹³C₁₂-T4 internal standard in plasma extractions was: 99?±?1.8 % in rainbow trout, 85?±?11 % in fathead minnow, 73?±?5.0 % in mummichog, 73?±?1.7 % in sockeye salmon, and 80?±?8.4 % in coho salmon. While absolute levels of thyroid hormones measured in identical plasma samples by LC/MS/MS and RIA varied depending on the assay used, T4/T3 ratios were generally consistent across both techniques. Less variability was measured among samples subjected to LC/MS/MS suggesting a more precise estimate of thyroid hormone homeostasis in the species targeted. Overall, a sensitive and reproducible method was established that takes advantage of LC/MS/MS techniques to rapidly measure TT4 and TT3 with negligible interferences in low volumes of plasma across a variety of teleost fishes.     

Simultaneous Nonchiral Determination of Artemisinin and Arteannuin B in Artemisia annua Using Circular Dichroism Detection

A new method was performed using on-line coupling of nonchiral reversed-phase liquid chromatography (RP-LC) to circular dichroism (CD) spectroscopy for simultaneous determination of artemisinin and arteannuin B in crude plant extracts of Artemisia annua. Analysis was carried out on an LC–CD system equipped with an Agilent TC-C₁₈ column (4.6 mm I.D. × 200 mm L, 5 μm) using gradient of acetonitrile. The method was validated to be practicable and reliable at alterable wavelength in the ranges of 220–420 nm as desired. LOD and LOQ of artemisinin and arteannuin B were 0.08, 0.26 and 0.31, 1.02 μg mL⁻¹, respectively. It was more sensitive than conventional LC–UV and comparatively cheaper than LC–MS in analysis of TCM.

     

Circadian variation and in vitro cytotoxic activity evaluation of volatile compounds from leaves of Piper regnellii (Miq) C. DC. var. regnellii (C. DC.) Yunck (Piperaceae)

Aiming detection of circadian variation in the chemical composition of volatiles from Piper regnellii, the leaves were collected during four different periods (8, 12, 16 and 20 h) in the same day. After extraction by hydrodistillation and GC/MS analysis, no significant variation was observed for the main compounds: germacrene D (45.6 ± 1.5–51.4 ± 3.1%), α–chamigrene (8.9 ± 1.3–11.3 ± 2.7%) and β–caryophyllene (8.2 ± 0.9–9.5 ± 0.3%). Evaluation of in vitro cytotoxicity against several cancer and non-tumourigenic cells indicated promising activity, especially to HeLa (human cervical carcinoma) with IC₅₀ ranging from 11 ± 3 to 17 ± 3 μg/mL. The obtained volatile oils were pooled and subjected to fractionation to afford pure β-caryophyllene, α-chamigrene and germacrene D, being this last compound the more active against HeLa cells with IC₅₀ of 7 ± 1 μg/mL (34 ± 5 μM). Therefore, the predominance of germacrene D in all analysed oils could justify, at least in part, the activity observed for the volatile compounds from P. regnellii leaves.     

LC–MS Method for the Sensitive Determination of Metoclopramide: Application to Rabbit Plasma, Gel Formulations and Pharmaceuticals

To support real biological sample application, a simple, selective and rapid LC–MS method has been developed and validated for the sensitive determination of metoclopramide in rabbit blood, ex vivo permeation studies and pharmaceutical dosage form. LC–MS analysis was performed isocratically on a Zorbax SB-C₁₈ column with a mobile phase consisting of methanol:ammonium acetate buffer (pH 3.0) 75:25 (v/v) at a flow rate of 0.70 mL min^?1. The outlet of the column was connected to a single quadrupole mass spectrometer with positive mass spectrometric detection. Ions were detected in the positive multiple reaction monitoring mode. The assay was linear over the concentration range of 1.25–200 pg μL^?1 with a limit of detection of 0.077 pg μL^?1 for standard solutions and 2.5–200 pg μL^?1 with a limit of detection of 0.42 pg μL^?1 for serum samples. The method is applicable, covering a variety of pharmaceutical and biological studies. Metoclopramide was extracted from rabbit blood by liquid–liquid extraction using ether as the extraction solvent. The reproducibility of the method was found to be between 0.96 and 1.98 % (RSD) values. The proposed method has been extensively validated for the determination of metoclopramide in all working media. The sample preparations, flow rate and run time of the analytical systems are not time consuming. Moreover, for the stability of metoclopramide, the effect of temperature, UV light, H₂O₂, HCl and NaOH were also investigated.     

New cytotoxic isoprenoid derivatives from the Red Sea soft coral Sarcophyton glaucum

Chemical investigation of the soft coral Sarcophyton glaucum collected from the Red Sea led to isolation of 11 isoprenoidal metabolites (1–11). A new sesquiterpenoid, 6-oxo-germacra-4(15),8,11-triene (1), a new natural cembranoid, sarcophinediol, along with two known sesquiterpenoids (2 and 3) and seven known cembranoids (5–11) was obtained. The structures of the compounds were established based on their NMR, MS, IR and UV spectral data. All compounds were evaluated for their cytotoxicity employing three cancer cell lines (HepG2, MCF-7 and HCT116). Compounds 4 and 6 showed significant cytotoxicity towards HepG2 with IC₅₀ values of 18.8 ± 0.07 and 19.9 ± 0.02 μM; respectively. Compounds 5–7 exhibited potent cytotoxicity against MCF-7 cells with IC₅₀ values of 9.9 ± 0.03, 2.4 ± 0.04 and 3.2 ± 0.02 μM, respectively. Compounds 1, 4 and 5 showed significant activities towards HCT116 cells with IC₅₀ values of 29.4 ± 0.03, 19.4 ± 0.02 and 25.8 ± 0.03 μM, respectively.     

LC–MS–MS Determination of Nikethamide in Human Plasma

A sensitive LC–MS–MS method with electrospray ionization has been developed for determination of nikethamide in human plasma. After addition of atropine as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 mm × 2.1 mm, 5 μm particle, Agilent Zorbax SB-C₁₈ column, with 45:55 (v/v) methanol–water containing 0.1% formic acid as mobile phase. LC–MS–MS was performed in multiple reaction monitoring mode using target fragment ions m/z 178.8 → 107.8 for nikethamide and m/z 289.9 → 123.8 for the internal standard. Calibration plots were linear over the range of 20.0–2,000 ng mL^?1. The lower limit of quantification was 20.0 ng mL^?1. Intra-day and inter-day precisions were better than 4.2 and 6.1%, respectively. Mean recovery of nikethamide from human plasma was in the range 65.3–71.1%.     

Determination of Pitavastatin in Human Plasma by LC–MS–MS

A simple and rapid LC–MS–MS assay was developed and validated for the quantitative determination of pitavastatin in human plasma. Sample pretreatment involved simple protein precipitation by addition of acetonitrile. Separation was on an Agilent 1.8 μm Zorbax SB-C18 column (150 mm × 4.6 mm) at 25 °C using isocratic elution with methanol–0.1% formic acid in water (85:15, v/v) at a flow rate of 0.4 mL min^?1. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the ion transitions m/z 422.0 → 290.1 for pitavastatin, and m/z 330.1 → 192.1 for paroxetine (IS). LC–MS–MS was found to improve the quantitation of pitavastatin in plasma and was successfully applied in pharmacokinetic studies.     

Simple Determination of 4-Methylimidazole in Soft Drinks by Headspace SPME GC–MS

A simple method to detect 4-methylimidazole in soft drinks is described. This method is based on headspace solid-phase micro-extraction and gas chromatography–mass spectrometry (HS-SPME GC–MS). The HS-SPME parameters (selection of fiber, extraction temperature, heating time, and pH) were optimized and selected. Under the established condition, the detection and the quantification limit were 1.9 and 6.0 μg L^?1 using 4 mL of the liquid sample, respectively. The relative standard deviation for five independent determinations at 100.0 and 500.0 μg L^?1 was less than 8 %. The calibration curve was y = 0.6027x–0.0033 with a linearity of r ² = 0.997. Using the proposed method, the levels of 4-MEI were detected in a range from 94.0 to 324.8 μg L^?1. The comparison of liquid chromatography tandem mass spectrometry (LC–MS/MS) with the proposed method was performed and the agreement with LC–MS/MS for all samples was acceptable.     

New macrocyclic diterpenes from Euphorbia connata Boiss. with cytotoxic activities on human breast cancer cell lines

Acetone:chloroform (1:2) extract of the aerial parts of Euphorbia connata Boiss. (Euphorbiaceae) was investigated for its diterpenoids. This led to the isolation of one known and two new diterpenes, belonging to the pentahydroxy-13(17)-epoxy-8,10(18)-myrsinadiene and tetrahydroxy-5,6-epoxy-14-oxo-jatropha-11(E)-ene classes. The structures were elucidated based on ¹³C and ¹H NMR as well as 2D NMR, IR and MS spectra and the cytotoxicity for compounds 1–3 were evaluated by using MTT assay against two human breast cancer cell lines. Myrsinane-type compounds – 3,7,14,15-tetraacetyl-5-propanoyl-13(17)-epoxy-8,10(18)-myrsinadiene (1) and 3,7,10,14,15-pentaacetyl-5-butanoyl-13,17-epoxy-8-myrsinene (2) – exhibited moderate inhibitory effects, with IC₅₀ values of 24.53 ± 3.39 and 26.67 ± 1.41 μM against the MDA-MB cell line, and 37.73 ± 3.41 and 34.57 ± 2.12 μM against the MCF-7 cell line, respectively. Jatrophane-type diterpene – 5,6-epoxy-8,9,15-triacetyl-3-benzoyl-14-oxo-jatropha-11(E)-ene (3) – showed weak cytotoxicity, with IC₅₀ values of 55.67 ± 7.09 μM against MDA-MB, and moderate cytotoxicity with IC₅₀ values of 24.33 ± 3.21 μM against MCF-7 cell line.     

LC–MS Determination and Pharmacokinetic Study of Salidroside in Rat Plasma after Oral Administration of Traditional Chinese Medicinal Preparation Rhodiola crenulata Extract

A simple, rapid and sensitive liquid chromatography–mass spectrometry (LC–MS) method was developed for the quantification of salidroside in rat plasma and the study of its pharmacokinetics after oral administration of 15 g kg^?1 Rhodiola crenulata extract to Wistar rats. A 200 μL plasma sample was extracted by acetonitrile and performed on Kromasil C₁₈ column (150 mm × 4.6 mm, 5 μm) with the mobile phase of acetonitrile–water (11:89) within a run time of 8 min. The analyte was monitored with electrospray ionization (ESI) by selected ion monitoring (SIM) mode. The target ions were m/z 299.20 for salidroside and m/z 150.00 for internal standard (IS) paracetamol. A good linear relationship was obtained over the range of 100–20,000 ng mL^?1 and the lower limit of quantification was 100 ng mL^?1. The validated method was successfully applied for the pharmacokinetic study of salidroside in rat. After oral administration of Rhodiola crenulata extract, the main pharmacokinetic parameters T _max, T _1/2, C _max, AUC _0?t and AUC _0?∞ were 0.56 ± 0.21 h, 7.91 ± 4.42 h, 3,386 ± 2,138 ng mL^?1, 16,146 ± 6,558 ng h mL^?1 and 18,599 ± 6,529 ng h mL^?1, respectively.     

Isolation and Identification of Antioxidative Peptides from Frog (Hylarana guentheri) Protein Hydrolysate by Consecutive Chromatography and Electrospray Ionization Mass Spectrometry

Frog (Hylarana guentheri) proteins were hydrolyzed by papain and Flavourzyme to obtain antioxidative peptides. The antioxidant activities of the frog protein hydrolysates (FPHs) were measured, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC₅₀?=?9.94?±?0.13 mg/mL), reducing power (0.39?±?0.01 at 5.0 mg/mL), and oxygen radical absorbance capacity (ORAC) value (789.15?±?75.10 μmol Trolox equivalents/g). The hydrolysates were purified by ultrafiltration, ion exchange chromatography, gel filtration chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). Through analysis of ESI-MS/MS, two dipeptides were identified as Leu/Ile-Lys (259.1607 Da) and Phe-Lys (293.1446 Da), respectively.     

Hepatoprotective and neuroprotective tocopherol analogues isolated from the peels of Citrus unshiu Marcovich

Three tocopherol analogues methoxytocopherol (1), α-tocopherol (2) and γ-tocopherol (3) were isolated from the peels of Citrus unshiu Marcovich. The protective effects of the isolated compounds against tert-butyl hydroperoxide-induced hepatotoxicity in human liver-derived HepG2 cells and glutamate-induced oxidative stress in HT22-immortalised hippocampal cells were evaluated. Compounds 1–3 were significantly protective in HepG2 cells with EC₅₀ values of 21.22 ± 2.01, 25.21 ± 2.11 and 25.25 ± 1.21 μM, respectively, and in HT22 cells, compounds 1–3 had EC₅₀ values of 20.62 ± 1.36, 6.44 ± 1.65 and 9.52 ± 1.54 μM, respectively.     

Comparison of LC–UV, LC–ELSD and LC–MS Methods for the Determination of Sesquiterpenoids in Various Species of Artemisia

Simple and specific analytical methods for the quantitative determination of sesquiterpenoids from various species of Artemisia plant samples were developed. By LC–UV, LC–ELSD, the separation was achieved by reversed-phase chromatography on a C18 column with water and acetonitrile both containing 0.025% trifluoroacetic acid as the mobile phase. In the LC–MS system, trifluoroacetic acid was replaced by 0.1% formic acid. The wavelength used for quantification of sesquiterpenoids with a diode array detector was 205 nm. The limits of detection by LC–MS was found to be 5, 10, 25, 50, 50 ng mL^?1. The limits of detection by LC–UV and LC–ELSD were found to be 5.0, 3.0, 100, 100, 7.5 μg mL^?1, by LC–UV and 50, 25, 30, 100 and 75 μg mL^?1 by LC–ELSD. LC–mass spectrometry coupled with electrospray ionization (ESI) interface is described for the identification and quantification of sesquiterpenoids in various plant samples. This method involved the use of the [M + H]⁺ ions of sesquiterpenoids in the positive ion mode with extractive ion monitoring.     

Simultaneous determination of sulfonamides and metabolites in manure samples by one-step ultrasound/microwave-assisted solid–liquid–solid dispersive extraction and liquid chromatography–mass spectrometry

An in-line matrix cleanup method was used for the simultaneous extraction of 15 sulfonamides and two metabolites from manure samples. The ultrasound/microwave-assisted extraction (UMAE) combined with solid–liquid–solid dispersive extraction (SLSDE) procedure provides a simple sample preparation approach for the processing of manure samples, in which the extraction and cleanup are integrated into one step. Ultrasonic irradiation power, extraction temperature, extraction time, and extraction solvent, which could influence the UMAE efficiency, were investigated. C₁₈ was used as the adsorbent to reduce the effects of interfering components during the extraction procedure. The extracts were concentrated, and the analytes were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) without any further cleanup. The isotopically labeled compounds sulfamethoxazole-d ₄, sulfamethazine-d ₄, sulfamonomethoxine-d ₄, and sulfadimethoxine-d ₆ were selected as internal standards to minimize the matrix effect in this method. The recoveries of the antibiotics tested ranged from 71 to 118 % at the three spiking levels examined (20, 200, and 500 μg?·?kg^-1). The limits of detections were 1.2–3.6 μg?·?kg^-1 and the limits of quantification were 4.0–12.3 μg?·?kg^-1 for the sulfonamides and their metabolites. The applicability of the method was demonstrated by analyzing 30 commercial manure samples. The results indicated that UMAE–SLSDE combined with LC–MS/MS is a simple, rapid, and environmentally friendly method for the analysis of sulfonamides and their metabolites in manure, and it could provide the basis for a risk assessment of the antibiotics in agricultural environments.     

LC–MS–MS Separation and Simultaneous Determination of Tolterodine and its Active Metabolite, 5-Hydroxymethyl Tolterodine in Human Plasma

A selective, sensitive and high throughput LC–MS–MS method has been developed and validated for the chromatographic separation and quantitation of tolterodine (TOL) and its metabolite 5-hydroxymethyl TOL in human plasma. Sample clean-up concerned liquid–liquid extraction of the drug, metabolite and their respective labelled internal standards from 300 μL human plasma. Both the analytes were chromatographically separated on a Symmetry C18 (100 mm × 4.6 mm, 5 μm particle size) analytical column using 10 mM ammonium formate (pH 5.0 ± 0.1, adjusted with formic acid) and acetonitrile (35:65, v/v) as the mobile phase with a resolution factor of 2.72. The method was validated over the concentration range of 0.025–10.0 ng mL^?1 for both analytes. The process efficiency found for TOL and its metabolite was 98.3 and 99.5%, respectively. The method was successfully applied to a pivotal bioequivalence study in 41 healthy human subjects after oral administration of a 2 mg tablet formulation under fasting conditions.     

LC–MS–MS Simultaneous Determination of l-Dopa and Its Prodrug l-Dopa n-Pentyl Ester Hydrochloride in Rat Plasma

A sensitive, simple and rapid LC–MS–MS method has been developed and validated for the simultaneous determination of l-dopa and l-dopa n-pentyl ester hydrochloride in rat plasma in the present study. The analytes were separated on a C₁₈ column (5 μm, 2.1 × 150 mm) with a security guard C₁₈ column (5 μm, 4 × 20 mm) and a triple-quadrupole mass spectrometer was applied for detection. The method was linear over the concentration ranges of 25–5,000 ng mL^?1 for l-dopa and 12.5–2,500 ng mL^?1 for l-dopa n-pentyl ester hydrochloride. Finally, the method was successfully applied to support the pharmacokinetic study.     

Effect of Salts (NaCl and Na2CO3) on Callus and Suspension Culture of Stevia rebaudiana for Steviol glycoside Production

Steviol glycosides are natural non-caloric sweeteners which are extracted from the leaves of Stevia rebaudiana plant. Present study deals the effect of salts (NaCl and Na₂CO₃) on callus and suspension culture of Stevia plant for steviol glycoside (SGs) production. Yellow-green and compact calli obtained from in vitro raised Stevia leaves sub-cultured on MS medium supplemented with 2.0 mg l^?1 NAA and different concentrations of NaCl (0.05–0.20 %) and Na₂CO₃ (0.0125–0.10 %) for 2 weeks, and incubated at 24?±?1 °C and 22.4 μmol m^?2 s^?1 light intensity provided by white fluorescent tubes for 16 h. Callus and suspension biomass cultured on salts showed less growth as well as browning of medium when compared with control. Quantification of SGs content in callus culture (collected on 15th day) and suspension cultures (collected at 10th and 15th days) treated with and without salts were analyzed by HPLC. It was found that abiotic stress induced by the salts increased the concentration of SGs significantly. In callus, the quantity of SGs got increased from 0.27 (control) to 1.43 and 1.57 % with 0.10 % NaCl, and 0.025 % Na₂CO₃, respectively. However, in case of suspension culture, the same concentrations of NaCl and Na₂CO₃ enhanced the SGs content from 1.36 (control) to 2.61 and 5.14 %, respectively, on the 10th day.