High performance liquid internal standards

Commercial grades of PVP, K-15, K-30, K-90, and K-120 and the quaternized copolymer of vinylpyrrolidone and dimthylaminoethylmethacrylate (poly-VP/ DMAEMA) made by International Specialty Products (ISP) were used in this study. PEO standard calibration kits were purchased from Polymer Laboratories Ltd. (PL), American Polymer Standards Corporation (APSC), Polymer Standards Service (PSS), and Tosoh Corporation (TSK). In addition, two narrow NIST standards, 1923 and 1924, were used to evaluate commercial PEO standards. Deionized, filtered water, and high-performance liquid chromatography grade methanol purchased from Aldrich or Fischer Scientific were used in this study. Lithium nitrate (LiN03) from Aldrich was the salt added to the mobile phases to control for polyelectrolyte effects. 

High performance liquid chromatography is used for the separation and quantitative analysis of a wide variety of mixtures, especially those in which the components are insufficiently volatile and/or thermally stable to be separated by gas chromatography. This is illustrated by the following method which may be used for the quantitative determination of aspirin and caffeine in the common analgesic tablets, using phenacetin as internal standard where APC tablets are available the phenacetin can also be determined by this procedure. 

D2O = deutered water. HPLC = high performance liquid chromatography. IS = internal standard. MeOH = methanol. MS = mass spectrometry. NMR = nuclear magnetic resonance. PDA = photodiode array detector. TEA = triethylamine. MTBE = methyl tert-butyl ether. 

Alhaique et al. [62] used a reversed phase high performance liquid chromatography method for the determination of miconazole in bulk or pharmaceuticals using bezafibrate as internal standard. 

Hasegawa et al. [76] measured miconazole serum concentration by a high performance liquid chromatographic method. The authors assessed whether the internal standard method produced an intra-assay error and found that the method gave more precise and more reproducible results compared to the absorption calibration curve method. With 0.5 pg/mL of miconazole, the coefficient of variation produced by that method was 3.41%, whereas that of the absorption calibration curve method was 5.20%. The concentration of absorptions calibration curve method showed higher values than the internal standard method. This indicated that the internal standard method was far more precise in measuring the miconazole serum concentrations than the absorption calibration curve method. 

Clark et al. [81] determined the time course of A-acetylation of primaquine by Streptomyces roseochromogenous and Streptomyces rimosus by quantitative high performance liquid chromatographic analyses of the culture broths. The A-5-bistri-fluoroacetyl derivative of primaquine was used as an internal standard in the analysis for the quantitation of primaquine A-acetate in microbial culture broths. S. roseochromogenous forms the highest level of primaquine A-acetate at 24—36 h after substrate addition, while S. rimosus is slower in its acetylation, peaking at 3 days after substrate addition. The formation of a novel dimeric compound from the reaction of primaquine with 8-(4-phthalimido-l-methylbutylamino)-6-methoxy quinoline is also reported. 

Rao et al. [87] developed a high performance liquid chromatographic method for the determination of primaquine phosphate in pharmaceutical dosage form. A /(-Bondapak NH2 column with chloroform-methanol (60 40) as eluent was selected with sulfalene as internal standard. The method was convenient with recovery of approximately 100% for primaquine. 

Dean et al. [93] used a high performance liquid chromatographic method for the simultaneous determination of primaquine and carboxyprimaquine in plasma with electrochemical detection. After the addition of the internal standard, plasma was deproteinized by the addition of acetonitrile. Nitrogen-dried supernatants, resuspended in mobile phase were analyzed on a C8 reversed-phase column. Limits of detection for primaquine and carboxyprimaquine were 2 and 5 ng/mL with quantitation limits of 5 and 20 ng/mL, respectively. The assay sensitivity and specificity are sufficient to permit quantitation of the drug in plasma for pharmacokinetics following low dose (30 mg, base) oral administration of primaquine, typically used in the treatment of malaria and P. carinii pneumonia. 

High performance liquid chromatography is used to determine the purity of calcitriol, and to separate it from related compounds. Using a 10 micron silica column of 25 cm length, and a mobile phase of spectroquality heptane ethyl acetate. methanol (50 50 1) at a flow rate of 1.7 ml/ minute, separation and quantitation are achieved. p-Dimethyl-aminobenzaldehyde may be used as an internal standard to compensate for variations in injection technique and instrumental conditions. With a 254 nm ultraviolet absorbance detector, 0.01 ug of calcitriol may be detected (3). 

The amount of cresol in the concentrated extract can then be determined by high performance liquid chromatography (HPLC) (DeRosa et al. 1987 Yoshikawa et al. 1986) or gas chromatography (GC) coupled to either a flame ionization detector (FID) or a mass spectrometer detection system (Angerer 1985 Needham et al. 1984). Separation of the cresol isomers by gas chromatography is readily accomplished, and the use of an appropriate internal standard allows the determination of their concentrations. Although exact detection limits were not given for the above GC methods, a concentration of 10 ppm appears to be readily determined. 

Determination of brinzolamide and its 3 principal metabolites (the N-desethyl, A -desmethoxypropyl and O-desmethyl analogs) in whole blood and plasma from clinical and pre-clinical studies was performed using high performance liquid chromatography (HPLC) with UV detection. After addition of a known amount of internal standard (AL-5138, the 4-methoxybutyl analog of brinzolamide), the sample was acidified with 50 mM sodium phosphate buffer, pH 3.0 and extracted with ethyl acetate. 

Rao et al. reported a high performance liquid chromatographic method to determine diloxanide furoate and metronidazole in single and in combined dosage forms [41]. A 30 mg equivalent of diloxanide furoate and 25 mg of metronidazole (either as the bulk drug substances or in powdered tablets) was dissolved in methanol, amidopyrine added as the internal standard, and the mixture analyzed by HPLC at room temperature. The analytical column (30 cm x 3.9 mm) consisted of p-Bondapak Cig, with 9 9 1 1 methanol water 0.05 M KH2PO4 0.05 M NaH2P04 as the mobile phase. The flow rate was 1 mL/min), and detection was performed at 254 nm. 

Fig. 2.2.9 High-performance liquid chromatography-tandem mass spectrometric analysis of AdoMet and AdoHcy. The analytes were evaluated by multiple reaction monitoring with the following transitions m/z 399 -> 250 for AdoMet m/z 402 -> 250 for the internal standard, tridenterated AdoMet (AdoMet +3) m/z 385 -> 135 for AdoHcy m/z 390 ->- 135 for the internal standard, pentadenterated AdoHcy (AdoHcy+5). Mass spectrometric conditions are described in the text. TIC total ion current, SRM selected reaction monitoring (Figure courtesy of Dr. Ries Duran, Amsterdam)...

Ito M, Ikeda K, Suzuki Y, Tanaka K, Saito M (2002) An improved fluorometric high-performance liquid chromatography method for sialic acid determination an internal standard method and its application to sialic acid analysis of human apolipoprotein E. Anal Biochem 300 260-266... 

Shackleton CHL, Kletke C, Wudy S, Pratt JH (1990) Dehydroepiandrosterone sulfate quantification in serum using high-performance liquid chromatography/mass spectrometry and a deuterated internal standard a technique suitable for routine use or as a reference method. Steroids 55 472-478... 

A Cantafora, R Masella. Improved determination of individual molecular species of phosphatidylcholine in biological samples by high performance liquid chromatography with internal standards. J Chromatogr 593 139-146, 1992. 

H Hasegawa. Vitamin D determination using high-performance liquid chromatography with internal standard-redox mode electrochemical detection and its application to medical nutritional products. J Chromat 605 215-220, 1992. 

ZH Gao, RG Ackman. Determination of vitamin K, in canola oils by high performance liquid chromatography with menaquinone-4 as an internal standard. Food Res Int 28 61-69, 1995. 

One of the first reliable methods for determining the concentrations of PFAs in tissues was the ion pairing method of Hansen et al. [79] (Fig. 6). Concentrations of PFOS in tissues, such as liver and blood plasma have been measured using high-performance liquid chromatography (HPLC) with electrospray mass spectrometry [1], One half milliliter of serum, 5 xL of an internal standard (e.g., tetrahydroperflurooctane sulfonic acid), 1 mL of 0.5 M tetrabutyl... 

Figure 15.5 Separation of Voriconazole and an internal standard by using SEC-HPLC. Adapted from Journal of Chromatography, B 691, D.A. Stopher and R. Gage, Determination of a new antifungal agent, voriconazole, by multidimensional high-performance liquid chromatography with direct plasma injection onto a size exclusion column , pp. 441 -448, copyright 1997, with permission from Elsevier Science.

Abbreviations AM, amlodipine AT, atorvastatin DS, diclofenac sodium EZ, ezetimibe FB, fenofibrate HAT, hydroxy atorvastatin IS, internal standard o-HAT, ortfio-hydroxy atorvastatin p-HAT, para-hydroxy atorvastatin HPLC-ESI-MS, high-performance liquid chromatography with electrospray tandem mass spectrometry LV, lovastatin NA, nicotinic acid NB, novobiocin FV, pravastatin RV, rosuastatin SV, simvastatin RT, roxethromycin UPLC, ultra performance liquid chromatography. 

The relative contribution of sample preparation depends on the steps in the measurement process. For instance, typically two-thirds of the time in an analytical chromatographic procedure is spent on sample preparation. An example of the determination of olanzapine in serum by high-performance liquid chromatography/mass spectroscopy (HPLC-MS) illustrates this point [3], Here, samples were mixed with an internal standard and cleaned up in a... 

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