Ammonium acetate, resolution

A liquid chromatography-mass spectrometry (LC-MS) method that can quantitatively analyze urinar y normal and modified nucleosides in less than 30 min with a good resolution and sufficient sensitivity has been developed. Nineteen kinds of normal and modified nucleosides were determined in urine samples from 10 healthy persons and 18 breast cancer patients. Compounds were separ ated on a reverse phase Kromasil C18 column (2.1 mm I.D.) by isocratic elution mode using 20 mg/1 ammonium acetate - acetonitrile (97 3 % v/v) at 200 p.l/min. A higher sensitivity was obtained in positive atmospheric pressure chemical ionization mode APCI(-i-). 

Fig. 2-10. The effect of flow rate on the resolution of methylphenidate enantiomers on vancomycin CSP (250 x 4.6 mm). The mobile phase was methanol 1.0 % triethyl-ammonium acetate (95/5 v/v) pH 4.1 at ambient temperature (23 °C).

For most free amino acids and small peptides, a mixture of alcohol with water is a typical mobile phase composition in the reversed-phase mode for glycopeptide CSPs. For some bifunctional amino acids and most other compounds, however, aqueous buffer is usually necessary to enhance resolution. The types of buffers dictate the retention, efficiency and - to a lesser effect - selectivity of analytes. Tri-ethylammonium acetate and ammonium nitrate are the most effective buffer systems, while sodium citrate is also effective for the separation of profens on vancomycin CSP, and ammonium acetate is the most appropriate for LC/MS applications. 

The luciferin produces a blue oxidation product during its purification process. In the DEAE chromatography of luciferin, this blue compound is eluted before the fractions of luciferin. The fractions of the blue compound were combined and purified by HPLC on a column of Hamilton PRP-1 (7 x 300 mm) using methanol-water (8 2) containing 0.1% ammonium acetate. The purified blue compound showed absorption peaks at 234, 254, 315, 370, 410, 590 (shoulder) and 633 nm. High-resolution FAB mass spectrometry of this compound indicated a molecular formula of C l C Nai m/z 609.2672 (M - Na + 2H)+, and mlz 631.2524 (M + H)+]. These data, together with the HNMR spectral data, indicated the structure of the blue compound to be 8. 

The HPLC system used consisted of a 30 x 2 mm Luna CN column with linear gradient elution employing two mobile phases A and B (A, 90% H2O 10% acetonitrile B, 10% H2O 90% acetonittile) with both phases containing 5 mM ammonium acetate and 0.2% formic acid. The hnear gradient commenced with 50 50 A B increasing to 100% B after 1 min of the analysis this composition was maintained for 1 min before returning to 50 50 A B after 4 min. Positive-ion ionspray (pneumatically assisted electrospray) was used to obtain mass spectra, with the spectrometer operating at a resolution of 5000. 

Tsuji and Goetz24 developed a quantitative high performance liquid chromatographic method for separating and measuring erythromycins A, B, and C, their epimers and degradation products. This method uses a /iBondapak Ci 8 reverse column with acetonitrile-methanol-O.2m ammonium acetate-water (45 10 10 25) as solvent. The pH and composition of the mobile phase may be adjusted to optimize resolution and elution volume. The authors utilized the procedure on USP reference standard and report a relative standard deviation of 0.64%. 

When chromatographic resolution of species based on modifications located at the protein surface is desired, it may be advisable to use conditions that favor retention of native conformation.17 Here, the standard acidic conditions described in the preceding text may be inappropriate, and mobile phases buffered near neutrality may be required. Buffers based on ammonium acetate, ammonium bicarbonate, and triethylammonium phosphate may prove more useful in resolving polypeptide variants with differing posttranslational modifications, amino acid substitutions, or oxidation and deamidation products. The addition of more hydro-phobic ion-pairing agents may be needed to obtain polypeptide retention, and a variety of alkyl sulfonates and alkyl amines have been described for specific applications.17... 

In a recent study, chiral separations for pyrethroic acids, which are the chiral building blocks of synthetic pyrethroids and the primary metabolites of the acid part of these potent ester insecticides, have been developed [62], For example, a polar-organic mobile phase allowed the complete baseline resolution of all four stereoisomers of chrysanthemic acid (2,2-dimethyl-3-(2-methylprop-l-enyl)-cyclopropanecarboxylic acid) on a 0-9-(tcrt-butylcarbamoyl)quinine-based CSP(acjj = 1.20, oLtrans = 1-35, critical Rs = 3.03) (Figure 1,32a). This chiral acid is the precursor of pyrethroids like allethrin, phenothrin, resmethrin, and tetramethrin but not excreted as metabolite. The primary acid metabolite of these pyrethroids is chrysanthemum dicarboxylic acid (3-[(l )-2-carboxyprop-l-enyl]-2,2-dimethylcyclopropanecarboxylic acid) the stereoisomers of which could also be resolved with a reversed-phase eluent (acetonitrile— 30-mM ammonium acetate buffer 90 10, v/v pHa = 6.0) and employing an O-9-(2,6-diisopropylphenylcarbamoyl)quinine-based CSP ads = 1-09, atrans = 1-50,... 

FIGURE 2.20 Direct chromatographic resolution of a mixture of 3-[(methoxycarbonyl)-methylthio]-2-amino-propanoic acid (1) and 3-[(methoxycarbonyl)methylthio]-2-acetamido-propanoic acid (2). Column TE CSP (250 x 4.5 mm ID) eluent MeOH/ammonium acetate 20 mM 85/15 (v/v) flow-rate 1 mL/min T = 25°C UV detection at 220 nm. 

In 1987, Ken Setchell first described the method for the isolation of phytoestrogen in soy (Setchell et al., 1987). The phytoestrogens daidzein, genistein, coumestrol, formononetin, and biochanin-A were separated on a Cl8 reversed-phase column (Hypersil ODS) with methanol-0.1 M ammonium acetate buffer, pH 4.6 (60 40 v/v), as eluent. The retention and resolution were affected by buffer concentrations, pH type, and proportion of organic solvent in the mobile phase. Detection in the low picograms range was achieved with an electrochemical detector, and the compounds were positively identified by HPLC-thermospray mass spectrometry. 

The ammonium acetate and acetic acid systems are less resolutive and practical (not UV transparent) than the TFA and TEAP based systems. The former are generally used to generate the acetate salt of peptides. For example, a peptide fraction partially purified with the TEAP system will be reapplied onto an HPLC column after dilution with water (1 1). The column is washed with 1% AcOH and the peptide is eluted by increasing the concentration of MeCN. As another example, the peptide is partially purified using 0.1% TFA, then reapplied onto an HPLC column after dilution with water (1 1). The column is washed first with an ammonium acetate solution to eliminate all TFA, followed by the 0.5% AcOH system to eliminate the ammonium counterion. The peptide is displaced with increasing concentrations of MeCN. 

A validated method (No. 63) for the analysis of benzoic acid, sorbic and para-hydroxybenzoic acids in fruit juices has been published in the IFU handbook (Anon, 1995e). In this method the resolution takes place on a C8 reverse-phase column. With this solvent system (methanolic/ammonium acetate, pH 4.55) sorbic and benzoic acids elute quite quickly and the less polar para-hydroxybenzoate esters elute much later. The method does stipulate that caution has to be taken by orange juice due to possible interferences by natural materials in the juice that elute close to benzoic acid. As sorbic and benzoic acids elute quite early in the chromatogram using this method, it would... 

Another study recorded the effect of the concentration of ammonium acetate on the chiral resolution of metomidate and etomidate on a Chiralpak AD-R column.. An increase in k values was observed with an increase in ammonium acetate concentration. On the other hand, the values of a decreased with an increase in ammonium acetate concentration [9]. The same trend was also observed on Chiralcel OD-R CSP for the chiral resolution of propranolol and trimepramine racemates [9]. 

FIGURE 5 Effect of the concentration of acetonitrile on (a) the retention (k) and the (b) separation (a) factors for the chiral resolution of (O) dansyl asparagine and ( ) dansyl valine on a Chiralpak WH column using 0.25 M ammonium acetate as the major component of the mobile phase. (From Ref. 53.)... 

The concentration of buffer is also a very important aspect in the optimization of the chiral resolution on these CSPs. It has been reported that an increase in buffer concentration caused a decrease in the retention and selectivity for all amino acids except for the basic amino acids. Therefore, the separation of basic amino acids is possible only with the most concentrated buffers. The buffers of concentrations in the 25-50-mM range were used for the chiral resolutions with some exceptions. In spite of this, few reports are available for the optimization of the chiral resolution by varying the ionic strength of the mobile phase. The effect of ionic strength of phosphate buffer on the chiral resolution of serine was carried out by Gubitz and Jellen [18] and the best resolution was achieved at 0.01 M concentration (Fig. 7). In another study, the concentration of ammonium acetate (0.001-0.01 M) was varied to optimize the chiral resolution of amino acids [19]. The effect of the concentration of ammonium acetate on the chiral resolution of amino... 

TABLE 5 Effect of the Concentrations of Ammonium Acetate on the Chiral Resolution of Amino Acids... 

FIGURE 2 Effect of temperature on the chiral resolution of dansyl valine amino acids on CSP I (see Fig. 1) using methanol-0.1 M ammonium acetate (80 20, v/v). (From Ref. 8.)... 

FIGURE 3 Chromatograms of the chiral resolution of (I) DNZ-leucine, (II) DNZ-glutamine, and (III) DNZ-phenylalanine on tert-butyl carbamoylated quinine CSP using (a) ammonium acetate (1 mM, pH 5.45) and (b) acetonitrile containing 1% ammonium acetate with gradient elution (DNZ 3,5-dinitrobenzyloxycarbonyl). (From Ref. 2.)... 

FIGURE 5 Effect of methanol concentration on the chiral resolution of ergot alkaloid on terguride-based CSP using 0.05 M ammonium acetate as the mobile phase at pH 7.2 (A),... 

Sivasubramanian and Anilkumar [81] described a simple reversed-phase HPLC method for the determination of omeprazole and domperi-done from tablet formulations. The analysis was carried out on a Hypersil ODS Ci8 (15 cm x 4.6 mm, 5 /jm) column using a mobile phase of methanol- 0.1 M ammonium acetate, pH 4.9 (60 40). The flow-rate and rim time were 1 ml/min and 10 min, respectively. The eluent was monitored at 280 nm. The method was reproducible, with good resolution between omeprazole and domperidone. The detector response was linear in the concentration range of 10-60 /[Pg.222]

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