Urine calibration curve

It is appropriate at this juncture to illustrate the power of chemiluminescence in an analytical assay by comparing the limits of sensitivity of the fluorescence-based and the chemllumlnescence-based detection for analytes in a biological matrix. The quantitation of norepinephrine and dopamine in urine samples will serve as an illustrative example. Dopamine, norepinephrine, and 3,4-dihydroxybenzy-lamine (an internal standard) were derivatized with NDA/CN, and chemiluminescence was used to monitor the chromatography and determine a calibration curve (Figure 15). The limits of detection were determined to be less than 1 fmol injected. A typical chromatogram is shown in Figure 16. 

Because of the possibility that the herbicide alachlor could adulterate food if either poultry or livestock consumed contaminated materials, Lehotay and Miller evaluated three commercial immunoassays in milk and urine samples from a cow dosed with alachlor. They found that milk samples needed to be diluted with appropriate solvents (1 2, v/v) to eliminate the matrix effect. One assay kit (selected based on cost) was also evaluated for use with eggs and liver samples from chickens. Egg and liver samples were blended with acetonitrile, filtered, and diluted with water. Linear calibration curves prepared from fortified egg and liver samples were identical... 

A volume equivalent to 0.25 mg of creatinine from 500 mL of urine of a healthy adult donor is analyzed as described above. The collection is then spiked with up to ten organic acids (1 mg/ml stock solutions), selected from those with an active calibration curve by GC-MS TIC, to mimic a concentration of 100 pg acid/mg creatinine. The spiked collection is aliquoted into screw-cap vials during continued mixing, then stored frozen. For GC-MS SIM analysis, all compounds listed in Table 3.1.5 are included in the abnormal control, at a concentration matching the fourth point of the calibration curve (1 1 molar ratio to the labeled internal standard, see Table 3.1.5). 

A calibration curve is recorded for each batch of samples (e.g., 100 pi, 50 pi, 25 pi, and 0 pi standard solution). The curve is plotted and results are calculated. Urines with absorptions exceeding the highest standard are diluted and remeasured. 

For internal quality control, urine from a known MPS patient and a solution containing chondroitin sulfate (50 pg/ml) are used. Results for urine and the artificial control should not exceed 3 standard deviations when plotted over time if it does, the test has to be repeated. In addition, absolute absorption values, the slope of the calibration curve, and the coefficient of variance (CV) of duplicates have to be within certain limits defined by the laboratory. 

Calibration curves for all polyols are prepared from a standard solution and are included in all series. The concentration ranges of the calibration curves are (based upon the concentrations found in urines) 0-4 nmol for ribitol, sedoheptitol and perseitol,... 

For quality control, 100 pi pooled urine spiked with 8 pmol/1 sedoheptitol and 8 pmol/1 perseitol is included in each series. In addition, a 4 x dilution of this sample and a pooled urine spiked with 180 pmol/1 erythritol, 90 pmol/1 threitol, 218 pmol/1 arabitol, 36 pmol/1 xylitol, 18 pmol/1 ribitol, 36 pmol/1 sorbitol, 145 pmol/1 mannitol, 55 pmol/1 galactitol, 16 pmol/1 sedoheptitol and 16 pmol/1 perseitol are included in each series. These three urine samples were chosen to obtain concentrations in the low, middle and high part of the calibration curves. 

QUANTITATIVE ASPECTS. Normally the presence of high quantities of the organic acid is sufficient for diagnosis. However, quantification is sometimes required. To do this, serial amounts of the individual acid must be carried through the derivatization, and a calibration curve set up. The various acids show widely different responses to the argon detector. From the standard curves the amounts can be derived by interpolation. Calculation on the basis of urine dilution must be included as well as the aliquot of the final solution that was taken for injection. The procedure for this is similar to that described for estriol in pregnancy urine. 

QUANTIFICATION. The addition of the internal standard (10 yg) to the urine before extraction is necessary for the quantification procedures. Known amounts of amphetamine and diphenylamine in the range 0-5 yg are chromatographed and calibration curves are plotted for peak area (y axis) versus concentration (x axis). 

Univariate and multivariate spectroscopy was applied to the analysis of spironolactone in presence of chlorthalidone [17]. Satisfactory results were obtained by partial least squares regression, with the calibration curve being linear over the range 2.92 -14.6 pg/mL. A kinetic-spectrophotometric method was described for the determination of spironolactone and canrenone in urine that also used a partial least-squares regression method [18]. After the compounds were extracted from urine, the spectra were recorded at 400 - 520 nm for 10 minutes at 30 second intervals. The relative error was less than 5%. 

A plasma calibration curve for ll-nor-A9-THC-9-carboxylic acid, 5a, is shown in Figure 9. There was reasonable linearity from 1.0-50 ng/ml plasma with detection limits of 0.5 ng or less per ml. Figure 10 presents similar data for a urine calibration curve. The method showed reasonable linearity between 2.0-100 ng/ ml urine. Figure 11 presents pharmacokinetic data. for plasma levels of a human volunteer, BS, over a 0.5 hour to 48 hour period comparing A9-THC and 11-nor acid levels after a dose of 5.0 mg of A9-THC by the intravenous route. Both parent compound and acid metabolite exhibited a biphasic elimination pattern although the levels of the acid did not fall as rapidly as parent compound. Elimination of the acid metabolite 5a in urine is shown in Figure 12. It is evident that urinary elimination proceeded rapidly as 80% of the total 11-nor-acid excreted was eliminated in the urine during... 

Shinozuka et al. [91] developed a sensitive method for the determination of four anthranilic acid derivatives (diclofenac sodium, aluminium flufenamate, mefenamic and tolfenamic acids) by HPLC procedure. The four drugs were converted into methylphthalimide (MPI) derivatives in a constant yield by reaction with /V-chloromethylphthalimide at 60°C for 30 min. The production of the MPI derivatives were confirmed by mass spectrometry. The MPI derivatives of the four drugs were separated by HPLC using a C-18 bonded phase LiChrospher RP-18 column (250 x 4 mm i.d.) with acetonitrile-water (80 20, v/v) as mobile phase. The flow rate was 0.8 mL/min. The UV absorbance was measured at 282 nm. The calibration curves of the MPI derivatives of the drugs were linear from 1.0 to 5.0 pg/rnL. The detection limits of the four drugs were 0.5-5 ng. The extraction procedure for the four anthranilic acid derivatives added in the plasma and urine was performed by using Extrelut 1 column. Yields of column extraction of 100 pL of plasma and urine samples (containing 0.5 pg of anthranilic acid derivatives) with 6 mL of ethyl acetate were 84-106%. 

Method. To 10 ml of the urine sample add 0.1 ml of nitric acid and allow to stand for 1 hour. Transfer diree 1-ml portions of die solution to separate test-tubes and to two of diem add, respectively, 1 ml of standard solutions containing 0.02 and 0.05 jag/ml of Cr to die third tube add 1 ml of 0.01 M nitric acid. Inhoduce 5 ]al of each solution into die graphite furnace, dry, ash, atomise, and record the absorbance at 357.9 nm. Record the absorbance of each of the diluted standard solutions in a similar manner. Plot die absorbance of each standard solution against die concentration of chromium, read off the concenhation in die three sample tubes, and calculate the concentration in the sample. The calibration curve should be linear at least in die range 0 to 0.03 lag/ml. 

Eluent. Hexane tetrahydrofuran (4 1) to which has been added 20 mg/litre of acetic acid. Calculate the content of phenylbutazone from a calibration curve prepared by repeating the procedure using appropriate reference solutions of phenylbutazone in plasma or urine. 

Inductively coupled plasma-atomic emission spectrometry was investigated for simultaneous multielement determinations in human urine. Emission intensities of constant, added amounts of internal reference elements were used to compensate for variations in nebulization efficiency. Spectral background and stray-light contributions were measured, and their effects were eliminated with a minicomputer-con-trolled background correction scheme. Analyte concentrations were determined by the method of additions and by reference to analytical calibration curves. Internal reference and background correction techniques provided significant improvements in accuracy. However, with the simple sample preparation procedure that was used, lack of sufficient detecting power prevented quantitative determination of normal levels of many trace elements in urine. 

Figures 8 and 9, on the other hand, show typical analytical calibration curves for the same sample materials when the net intensity ratio (net analyte line intensity/net internal reference line intensity) was used as the measure of response. In this case, the cmrves exhibited a considerably smaller range of slopes for the various urine samples and were in much better agreement with the data for the reference solutions.

Figure 6, Analytical calibration curves for iron in 1% NaCl reference solutions (O) and in the dilute (D), normal (N)y and concentrated (C) urine samples. See Table IV, The analysis wavelength was 261,2 nm.

Figure 8. Analytical calibration curves for iron as in Figure 6, Here, however, the analyte responses are in terms of the ratio of the net intensity of the analytical line (Fe 261,1 nm) to the net intensity of an internal reference element line (Y 371.0 nm). Although they are shown in the figure, the data points for the dilute, normal, and concentrated urine samples are not readily distinguishable.

Figure 11, Analytical calibration curves for zinc in urine with yttrium as the internal reference element. The analytical and internal reference line wavelengths were 213,9 and 371.0 nm, respectively. (X) Additions to the urine samples, (O) additions to the 1% NaCl reference soltuions.

The analysis of MPH from human urine was used as a model to demonstrate the direct-infusion quantitative bioanalysis from a polymer-based microfluidic chip electrospray emitter [62]. A calibration curve in the range 0.4-800 ng/ml was acquired. 

By means of this CD-enhanced fluorescence method, the authors could determine 11-MeBPHT in spiked human urine samples [23]. The standard addition procedure, based on the addition of a constant volume of the spiked urine sample to solutions of increasing methyl-BPHT concentration, was applied using spectrofluorimetric measurements. The linear calibration curves and standard addition linear plots were parallel, which suggested the absence of matrix interferences in the urine samples and permitted the validation of the method. Good linearity and precision were also obtained for the standard addition plots, with correlation coefficients close to unity. Satisfactory recovery percentage values ranging from 97 to 108% were found in the urine samples for the determination of 11-MeBPHT concentrations at low xg/mL levels. 

Indapamide can be analyzed in blood, plasma and urine by HPLC and fluorescence methods. A specific and sensitive assay method for the analysis of indapamide in urine, blood and plasma was developed (31). The method uses a 250 x 4.6 mm i.d. Zorbax ODS (5 pm particle size) column and a mobile phase of 0.1 M sodium acetate buffer (pl =3.6)/acetonitrile at a ratio of 65/35 (v/v). This method uses an internal standard of sulfanilanilide. Calibration curves obtained by plotting the ratio of the peak height of indapamide to that of sulfanilanilide versus the concentration of indapamide were linear over the concentration ranges of 25-200 ng/mL for plasma and 50-400 ng/mL for blood and urine using UV detection at 241 nm. Another publication reports a high performance liquid chromatographic assay method for monitoring indapamide and Its major metabolite in urine (32). 

It is possible to determine the presence of testosterone - 100 times lower than the dehydro compounds - in 2 pi of urine, i.e., 100 pg of free testosterone mixed with 100 ng of the dehydroepiandrosterone can be detected. These assays were made possible by the use of a calibration curve [210]. 

A calibration curve for the colorimetric determination of phosphorous in urine is prepared by reacting standard solutions of phosphate with molybdenum(VI) and reducing the phosphomolybdic acid complex to produce the characteristic blue color. The measured absorbance A is plotted against the concentration of phosphorous. From the following data, determine the linear least-squares line and calculate the phosphorous concentration in the urine sample ... 

Using DCP-AES, Roberts and Williams (1990) determined silicon in serum and urine after simple dilution in a 1% nitric acid solution. In the absence of any spectral or background interference, samples could be measured against aqueous standards. Matrix-matched calibration curves were used by Bercowy et al. (1994) who diluted serum and urine samples in water. Jackson et al. (1998) treated serum samples with highly pure nitric acid (2%) followed by dilution with distilled de-ionized water. 

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