Internal standard graph

Internal standard of Rh with 2 ppb concentration was added to all the solutions. Three certified Multi-element solutions (CLMS-1,-2,-4, SPEX, USA) were employed for constmcting calibration graphs. 

The peak area of the unknown (Ax) relative to the peak area of the internal standard (A ) is obtained. Conversion of the measured ratio to a concentration is achieved by comparing it to area ratios of the solutions of known analyte concentration, to which the same quantity of internal standard has been added. A graph of the ratio of the peak area of the component to be measured (A ) to the peak area of the internal standard (Ais) versus the ratio of the weight of the component to be measured (W ) to the weight of the internal standard (Wu.) for the known solutions results in a graph from which the concentration of the component(s) in the unknown matrix (Ax) can be determined (Figure 3.1). 

In many cases when methods involve internal or external standards, the solutions used to construct the calibration graph are made up in pure solvents and the signal intensities obtained will not reflect any interaction of the analyte and internal standard with the matrix found in unknown samples or the effect that the matrix may have on the performance of the mass spectrometer. One way of overcoming this is to make up the calibration standards in solutions thought to reflect the matrix in which the samples are found. The major limitation of this is that the composition of the matrix may well vary widely and there can be no guarantee that the matrix effects found in the sample to be determined are identical to those in the calibration standards. 

Calibration of an internal standard method is done by preparing standard samples of varying concentration. The same amount of IS is added to each, and the standard samples are analyzed using a developed method. The detector response, area or height, of each standard is determined, and a ratio is calculated. The graph of concentration vs. area ratio is plotted. The method is considered linear if the correlation coefficient is 0.99 or better. The response factor RF is calculated as... 

The improvements to the first three steps of scheme 1 were accomplished using GC as a major analytical tool. A capillary GC internal standard method, described above, was used to monitor the first three steps of scheme 1. Figure 10 is a typical chromatogram of the internal standard method for step 1 of scheme 1. To follow a reaction, a known amount of internal standard was added to the reaction vessel. Aliquots were withdrawn at intervals and analyzed on GC. A graph of yield vs. reaction time was prepared to determine the optimum time for completion of the reaction. 

Theobromine was determined by GC in various foods (bitter chocolate, milk chocolate, chocolate cake, cocoa powder, chocolate milk), and results are given in graphs and tables.27 Homogenized samples were boiled in alkaline aqueous media, then fat was extracted with n-hexane. The aqueous layer was acidified with diluted HC1 and NaCl was added. Theobromine was extracted from this treated aqueous solution with dichloromethane and the extract was evaporated to dryness. The residue was redissolved in dichloromethane containing an internal standard. GC analysis was performed on a column packed with 1% cyclohexane dimethanol succinate on Gaschrom Q, with FID. Average recoveries were 99 to 101%, coefficient of variation was less than 3% and the limit of detection for theobromine in foods was about 0.005%. 

Szathmary and Luhmann [50] described a sensitive and automated gas chromatographic method for the determination of miconazole in plasma samples. Plasma was mixed with internal standard l-[2,4-dichloro-2-(2,3,4-trichlorobenzyloxy) phenethyl]imidazole and 0.1 M sodium hydroxide and extracted with heptane-isoamyl alcohol (197 3) and the drug was back-extracted with 0.05 M sulfuric acid. The aqueous phase was adjusted to pH 10 and extracted with an identical organic phase, which was evaporated to dryness. The residue was dissolved in isopropanol and subjected to gas chromatography on a column (12 m x 0.2 mm) of OV-1 (0.1 pm) at 265 °C, with nitrogen phosphorous detection. Recovery of miconazole was 85% and the calibration graph was rectilinear for 0.25 250 ng/mL. 

Gotti et al. [42] reported an analytical study of penicillamine in pharmaceuticals by capillary zone electrophoresis. Dispersions of the drug (0.4 mg/mL for the determination of (/q-penicillaminc in water containing 0.03% of the internal standard, S -met hy I - r-cystei ne, were injected at 5 kPa for 10 seconds into the capillary (48.5 cm x 50 pm i.d., 40 cm to detector). Electrophoresis was carried out at 15 °C and 30 kV, with a pH 2.5 buffer of 50 mM potassium phosphate and detection at 200 rnn. Calibration graphs were linear for 0.2-0.6 pg/mL (detection limit = 90 pM). For a more sensitive determination of penicillamine, or for the separation of its enantiomers, a derivative was prepared. Solutions (0.5 mL, final concentration 20 pg/mL) in 10 mM phosphate buffer (pH 8) were mixed with 1 mL of methanolic 0.015% 1,1 -[ethylidenebis-(sulfonyl)]bis-benzene and, after 2 min, with 0.5 mL of pH 2.5 phosphate buffer. An internal standard (0.03% tryptophan, 0.15 mL) was added and aliquots were injected. With the same pH 2.5 buffer and detection at 220 nm, calibration graphs were linear for 9.3-37.2 pg/mL, with a detection limit of 2.5 pM. For the determination of small amounts of (L)-penicillamine impurity, the final analyte concentration was 75 pg/mL, the pH 2.5 buffer contained 5 mM beta-cyclodextrin and 30 mM (+)-camphor-10-sulfonic acid, with a voltage of 20 kV, and detection at 220 nm. Calibration graphs were linear for 0.5-2% of the toxic (L)-enantiomer, with a detection limit of 0.3%. 

A graph is plotted between the concentration values and the ratios obtained from the physical value (i.e., peak area of absorption) of the internal standard and the series of known concentrations, thereby producing a straight line. Any unknown concentration may be determined effectively by adding the same amount of internal standard and locating exactly where the ratio obtained falls on the concentration scale. 

Amantea and Narang [58] used a reversed-phase HPLC method for the quantitation of omeprazole and its metabolites. Plasma was mixed with the internal standard (the 5-methyl analog of omeprazole), dichlor-omethane, hexane, and 0.1 M carbonate buffer (pH 9.8). After centrifugation, the organic phase was evaporated to dryness and the residue was dissolved in the mobile phase [methanol-acetonitrile-0.025 M phosphate buffer of pH 7.4 (10 2 13)] and subjected to HPLC at 25 °C on a column (15 cm x 4.6 mm) of Beckman Ultrasphere C8 (5 ym) with a guard column (7 cm x 2.2 mm) of Pell C8 (30-40 /im). The mobile phase flow-rate was 1.1 ml/min with detection at 302 nm. The calibration graphs are linear for <200 ng/ml, and the limits of detection were 5, 10, and 7.5 ng/ml for omeprazole, its sulfone, and its sulfide, respectively. The corresponding recoveries were 96.42% and 96% and the coefficients of variation (n = 5 or 6) were 3.0-13.9%. 

Internal standard calibration can be used to compensate for variation in analyte recovery and absolute peak areas due to matrix effects and GC injection variability. Prior to the extraction, a known quantity of a known additional analyte is added to each sample and standard. This compound is called an internal standard. To prepare a calibration curve, shown in Figure 4.6b, the standards containing the internal standard are chromatographed. The peak areas of the analyte and internal standard are recorded. The ratio of areas of analyte to internal standard is plotted versus the concentrations of the known standards. For the analytes, this ratio is calculated and the actual analyte concentration is determined from the calibration graph. 

The resulting microdialysate samples were mixed (9 1) with acetyl-(3-methylcholine (internal standard, lng/pL), and a 950 pL portion was analyzed on a Discovery Cig column (50 cm x 2.1 mm i.d.). The method used 20 mM ammonium acetate/20mM heptafluorobutyric acid in methanol/water (1 9, pH 3.2) as the mobile phase at 0.4mL/min, and the analyte and internal standard were detected by tandem ion-trap mass spectrometry. The calibration graph was linear up to 500fg/pL of acetylcholine, and the on-column detection limit was 200 fg acetylcholine. 

Nimodipine was determined using a HPTLC method in tablets after their extraction with methanol (paracetamol was used as an internal standard). The plates were developed with toluene/ethylacetate (1 1), and densito-metric detection was effected at 240 nm. Calibration graphs were linear from 25 ng (the detection limit) to 200 ng. Recovery was 96.03%, and the relative standard deviation was 0.56% [13]. 

Nimodipine was determined in tablets by GC after extraction with chloroform (using nifedipine as an internal standard) [16]. A column (3 ft x 2 mm) of 10% of OV-1 on Chromosorb G (AW), 80-100 mesh treated with DMCS and operated at 225° C, was used with nitrogen as the carrier gas. Flame ionization was used as the means of detection. The calibration graph was linear over the range 0.5-2 ng/mL, and the coefficient of variation was 2.28. 

Nimodipine in plasma was determined by a GC method [17]. Plasma was treated with 2 M NaOH after addition of the internal standard, and then extracted with toluene. The column (10 m x 0.31 mm) consisted of cross-linked 5% phenylmethyl silicone, and the method used temperature programming from 90°C (held for 1 min) to 255°C at a heating rate of 25°C/min. Helium was used as a carrier gas, and the N-P detection mode was employed. The calibration graph was linear from 2 to 50 ng/mL, and the detection limit was 0.5 ng/mL. 

The stability of nimodipine injectable formulations was studied using reversed phase HPLC, which made use of methyltestosterone as an internal standard. A YWG C18 column was eluted with methanol/water (13 7, v/v), and detection was effected at 238 nm. The calibration graph was linear over the range of 5.98-299 pM/L [21],... 

Wang et al. studied the stability of nimodipine by HPLC, using beclomethazone dipropionate as an internal standard [33]. The method used a stainless steel column (25 cm x 4 mm) containing YWG C18H37, which was eluted at a flow rate of 1 mL/min) with methanol-water-ethyl ether (35 15 4). Detection was effected at 238 nm. The calibration graph... 

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