Liquid chromatography setup-dependent artefactual methionine oxidation of peptides: The importance of an adapted quality control process.

In both biologics quality control experiments and protein post-translational modification studies, the analytical system used is not supposed to bring any artefactual modifications which could impair the results. In this work, we investigated oxidation of methionine-containing peptides during reversed-phase (RP) chromatographic separation. We first used a synthetic methionine-containing peptide to evaluate this artefactual phenomenon and then considered more complex samples (i.e., plasma and HeLa protein digests). The methionine oxidation levels of the peptides were systematically assessed and compared for the long-term use of the analytical column, the sample trapping time, the gradient length, the sample load and the nature of the stationary phase (HSS T3 from Waters, YMC Triart C18 from YMC Europe GmbH and BEH130 C18 from Waters). In addition to the oxidation of methionine in solution, we observed on the HSS T3 and the BEH130 stationary phases an additional broad peak corresponding to an on-column oxidized species. Considering the HSS T3 phase, our results highlight that the on-column oxidation level significantly increases with the age of the analytical column and the gradient length and reaches 56 % when a 1-year-old column set is used with a 180 min-long LC method. These levels go to 0 % and 18 % for the YMC Triart C18 and the BEH130 C18 phases respectively. Interestingly, the on-column oxidation proportion decreases as the injected sample load increases suggesting the presence of a discrete number of oxidation sites within the stationary phase of the analytical column. Those findings observed in different laboratories using distinct set of columns, albeit to varying degrees, strengthen the need for a standard of methionine-containing peptide that could be used as a quality control to appraise the status of the liquid chromatographic columns.

Standardization of chromatographic signals - Part II: Expanding instrument-agnostic fingerprints to reverse phase liquid chromatography.

There is a large amount of literature relating to multivariate analytical methods using liquid chromatography together with multivariate chemometric/data mining methods in the food science field. Nevertheless, dating the obtained results cannot be compared as they are based on data acquired by a particular analytical instrument, thus they are instrument-dependant. Therefore, this creates difficulties in generating a database large enough to gather together all the variability of the samples. The solution to this problem is to obtain an instrument-agnostic chromatographic signal that is independent of the chromatographic state, i.e., measuring instrument or particular condition of the same instrument from which it was acquired. This paper describes the methodology to be followed to obtain standardized instrumental fingerprints when liquid chromatography is used for prior separation. For this purpose both internal and external chemical standards series are used as references. As an application example, we have applied this methodology for the determination of biophenols in olive oil by liquid chromatography coupled to ultraviolet-visible detector (LC-UV), using three different LC-UV instruments. The instrument-agnostic fingerprints obtained show a high grade of similarity, regardless of the state of the chromatographic system or the time of acquisition.

Importance of Particle Pore Size in Determining Retention and Selectivity in Reversed Phase Liquid Chromatography.

Column selection often centers on the identification of a stationary phase that increases resolution for a certain class of compounds. While gains in resolution are most affected by selectivity of the stationary phase or modifications of the mobile phase, enhancements can still be made with an intentional selection of the packing material's microstructure. Unrestricted mass transfer into the particle's porous structure minimizes band broadening associated with hindered access to stationary phase. Increased efficiency, especially when operating above the optimal flow rates, can be gained if the pore size is significantly larger than the solvated analyte. Less studied are the effects of reduced access to pores due to physical hindrance and its impact on retention. This article explores the relationship between pore size and reversed phase retention, and specifically looks at a series of particle architectures with reversed phase and size exclusion modes to study retention associated with access to stationary phase surface area.

Development of a RP-HPLC method for simultaneous determination of reference markers used for in-situ rat intestinal permeability studies.

One of the most common techniques for assessing the intestinal absorption characteristics of drugs is single-pass intestinal perfusion (SPIP) method. Metoprolol tartrate (MT, reference standard) and phenol red (PR, zero permeability marker) are the compounds that are normally used in SPIP studies. The aim of this study was to develop a reverse phase high-performance liquid chromatography (RP-HPLC) method combined with UV-detection for the simultaneous determination of MT and PR in the perfusion medium used in SPIP experiments. Elution was performed using a Restek Raptor C18 column (5 µm, 4.6 mm × 250) at a temperature of 25 °C. The mixture of the mobile phase consisted of (MeOH):(Phosphate buffer solution, PBS), (20 mM, pH 3.0 adjusted with ortho-phosphoric acid),(55:45, v/v). Flow rate and column temperature were set at 1.2 mL min-1 and 25 °C, respectively. MT and PR were injected as 20 µL into the HPLC system. UV detection was performed at 227 nm. The obtained retention times were reported as 2.89 and 3.80 min for MT and PR, respectively. The developed RP-HPLC method was validated according to Q2(R1) guideline of The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). The method was linear within the range of 2-50 µg mL-1 for PR and 10-75 µg mL-1 for MT. The developed RP-HPLC method was successfully applied on determination of MT and PR in perfusion medium. The developed method could be helpful for researchers working on in-situ rat intestinal permeability studies and it could be easily modified on further studies.

A validated RP-HPLC method for the determination of piperidone analogue of curcumin.

Curcumin (Diferuloylmethane) is a natural product extracted from the root of Curcuma longa. 5-Bis (4-hydroxy-3-methoxybenzylidene)-N-methyl-4-piperidone, the piperidone analogue of curcumin (PAC), was one of the analogues that, demonstrated potential anticancer effects against breast and colon cancers compared with native curcumin. A simple, accurate, and rapid isocratic reverse phase high performance liquid chromatography (HPLC) analytical method utilizing UV detection was developed and validated for the determination of PAC utilizing C18 column with run time was 7 min. Chromatogram showed a peak of PAC at retention time of 5.8±0.92 min. The method was validated for linearity, accuracy, precision, limit of detection, limit of quantitation and robustness. Linear relationship (r > 0.99) was observed between AUP of PAC and the corresponding concentrations over 100-10000µg/mL. The LOQ of this assay was 3.9ng/mL with a corresponding relative standard deviation of 4.8 and 4.0%. The LOD was 13.1ng/mL at a signal-to-noise ratio of >3.

Evaluation of selected pharmaceuticals and personal care products in water matrix using ion trap mass spectrometry: A simple weighted calibration curve approach.

A novel analytical method is presented for 12 target pharmaceutical and personal care products (PPCPs), belonging to different classes like antibiotics, non-steroid anti-inflammatory drugs, parabens, UV-filters, plasticizer, and antibacterials. The method development comprises of solid-phase extraction (SPE) with lipophilic-hydrophilic material balanced Oasis HLB cartridge, followed by reverse-phase liquid chromatography interfaced to linear ion trap tandem mass spectrometry (LC-MS/MS) with electrospray ionization. Chromatographic separation was achieved with a gradient elution of 25 min run time using 5 mM ammonium acetate buffer with pH adjustment using acetic acid. In addition, cost effective organic solvent with buffer used together as the mobile phase with Chromatopak C18 column (150 mm × 4 mm, 5-µm,) in negative ionization mode. Recoveries ranged from 61.74 % to 119.89 % for most of the compounds. Matrix-matched calibration curves were used for counterbalancing the matrix effects for all the analytes, and ibuprofen D3 internal standard was used for assessing the effectiveness of extraction technique and monitoring the recovery of sample analysis. Simple empirical weighted linear regression curve technique was adopted practically for each analysis in enhancing the analyte accuracy at lower quantification level. The 1/x2 model was selected as the best suitable model for quantification of analytes, which can be evaluated by deviation from back-calculated concentration in terms of percentage relative error (%RE). Weighted calibration curves with regression value for most of the compounds were ≥ 0.98, except triclosan with a regression value ≥ 0.93. Precision showed as % relative standard deviation (%RSD) were always below 15.0 %. Accuracy-test was evaluated by the statistical one-sample t-test and no significant difference was observed between nominal and experimental value. The limit of quantification (LOQ) ranged from 3.0 ng/mL (BP1) to 1000 ng/mL (naproxen). Finally, the validated method was used for the first time to determine target analytes in surface water samples collected from Arkavathi river flowing across southern India's Bengaluru city.

Evaluating MISER chromatography as a tool for characterizing HILIC column equilibration.

Hydrophilic Interaction Liquid Chromatography (HILIC) is a technique for retaining polar analytes that uses polar stationary phases and acetonitrile-rich mobile phases. While this technique has several advantages over reversed-phase liquid chromatography (RPLC), one main drawback is the reported need for longer column equilibration. The reason for this is not fully understood and is a topic of current investigation. In order to better understand and reduce the equilibration needs, accurate characterization of column equilibration under varying conditions is required. The current method of characterizing HILIC column equilibration produces limited data points per test, or low time resolution, and is highly dependent on the column and probe compounds being used. There is a need for an improved method for characterizing HILIC column equilibration, especially if trends across stationary phases are to be observed. In this work, MISER, or Multiple Injections in a Single Experimental Run, is evaluated as a possible tool for characterizing HILIC column equilibration. MISER improves time resolution by allowing for replicate injections without interruption of data collection, enabling a more thorough evaluation of column equilibration compared to traditional techniques. Experimental results gathered using MISER show that equilibration of a BEH Amide column is notably shorter when equilibrating from acetonitrile to mobile phases containing higher percentages of water. Column equilibration to a 10% aqueous mobile phase was found to be approximately 5-fold faster than equilibration to a 3% aqueous mobile phase.

Quantitative Structure-Retention Relationships with Non-Linear Programming for Prediction of Chromatographic Elution Order.

In this work, we employed a non-linear programming (NLP) approach via quantitative structure-retention relationships (QSRRs) modelling for prediction of elution order in reversed phase-liquid chromatography. With our rapid and efficient approach, error in prediction of retention time is sacrificed in favor of decreasing the error in elution order. Two case studies were evaluated: (i) analysis of 62 organic molecules on the Supelcosil LC-18 column; and (ii) analysis of 98 synthetic peptides on seven reversed phase-liquid chromatography (RP-LC) columns with varied gradients and column temperatures. On average across all the columns, all the chromatographic conditions and all the case studies, percentage root mean square error (%RMSE) of retention time exhibited a relative increase of 29.13%, while the %RMSE of elution order a relative decrease of 37.29%. Therefore, sacrificing %RMSE(tR) led to a considerable increase in the elution order predictive ability of the QSRR models across all the case studies. Results of our preliminary study show that the real value of the developed NLP-based method lies in its ability to easily obtain better-performing QSRR models that can accurately predict both retention time and elution order, even for complex mixtures, such as proteomics and metabolomics mixtures.

Stability kinetics of fusidic acid: Development and validation of stability indicating analytical method by employing Analytical Quality by Design approach in medicinal product(s).

A simple, rapid, accurate, reproducible and sensitive reverse phase HPLC method for the estimation of fusidic acid (FA) by means of Analytical Quality by Design (AQbD) was the aim of the present study. Initially, the vital pre-requisites for AQbD like analytical method target profile and critical analytical attributes (CAAs) like theoretical plates, tailing factor and percent assay were defined. An octadecyl silyl silica C18 column with a packing size of 5 µm was employed and the detection was performed at 235 nm using UV-detector. The separation was performed with isocratic elution employing mixture of methanol: acetonitrile (5: 95, v/v) and an aqueous phase with pH of 2.8 containing 0.1% orthophosphoric acid in the ratio of 60: 40 (v/v). Ishikawa fish-bone diagram provided the basis of the variation in CAAs with various inputs. Taguchi Design was selected as the initial screening design to select critical method parameters (CMPs) affecting method development and Central Composite Design (CCD) was further applied for systematic optimization of chromatographic method by evaluating CAAs. Crucial parameters viz. limit of detection, limit of quantification, specificity, linearity and sensitivity were employed to validate the method in accordance with the ICH guidelines. Stress degradation studies were performed and the developed method was able to successfully differentiate the degraded products from the parent drug, that too in a topical gel. In conclusion, the findings of the present study validated the utility of AQbD in the systematic design of a liquid chromatographic method with fine sensitivity for FA estimation in medicinal products.

Qualitative evaluation of high pH mass spectrometry-compatible reversed phase liquid chromatography for altered selectivity in separations of intact proteins.

Intact proteins are increasingly being recognized as potential biomarkers and biotherapeutic agents for cancer and other serious diseases. Low pH reversed phase plays an important role in both single and multidimensional protein separations for resolving complex protein samples prior to mass spectrometric detection. In this work, we evaluated the use of high pH reversed phase liquid chromatography as an alternative chromatographic separation to gain different selectivity while maintaining the high resolving power and MS compatibility of reversed phase separations. The altered selectivity gained by high pH reversed phase liquid chromatography can further help to separate unresolved protein peaks or to increase peak capacity and resolving power of a multidimensional setup for complex biological samples. Hence, we evaluated the use of different MS-friendly buffers, ion pairing reagents, and stationary phases (silica- and polymer-based) at alkaline pH for intact protein separations. The best chromatographic separation, with complementary selectivity to low pH reversed phase, was achieved using triethylammonium bicarbonate at pH 10 and hybrid silica particles.

A new MS compatible HPLC-UV method for Teicoplanin drug substance and related impurities, part 1: Development and validation studies.

Teicoplanin is a glycopeptide antibiotic prepared by fermentation from cultures of Actinoplanes teichomyceticus, used as drug of last resort for the treatment of bacterial infections in humans. This study, which is the first in a series of two parts, describes the development of a LC method for the separation of Teicoplanin drug substance and its related impurities compatible with MS detection. The separation conditions for Teicoplanin were set on a LiChrospher 100 RP-18 column under gradient elution with a mobile phase composed of ammonium formate 25 mM at pH 6.00 and ACN. The new method was shown equivalent in terms of selectivity to the one reported in the European Pharmacopoeia Teicoplanin monograph, and was validated according to ICH Q2 R1 guidelines for the drug substance assay. The new method offers similar performance to the compendial one but has the advantage of being fully compatible with MS and it can be proposed as a useful tool also for controlling the quality of Teicoplanin fermentation batches and the occurrence of potential impurities.

Development and validation of simple step protein precipitation UHPLC-MS/MS methods for quantitation of temozolomide in cancer patient plasma samples.

Temozolomide (TEMODAL™) (TMZ) is an antineoplastic agent that is primarily used for the treatment of glioblastoma and anaplastic gliomas, two aggressive forms of brain cancer. Due to the poor prognosis of brain tumour patients, there is an increasing body of research into improving the stability and delivery of TMZ past the blood brain barrier using carrier molecules. These require accurate determination of TMZ levels for biodistribution and pharmacokinetic evaluation. Unfortunately, current methodologies for the determination of TMZ in human plasma suffer from low reproducibility, recovery, sensitivity or cost ineffective procedures associated with extensive sample cleaning. To surpass these disadvantages, we developed two bioanalytical methods with high sensitivity and excellent recovery for the determination of TMZ in human plasma at minimum cost. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used and both methods were validated under US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) guidelines. The two methods had minor differences in the sample pre-treatment and each method was developed and applied in separate laboratories. Theophylline was selected as internal standard (IS). Calibration curves were linear over the range of 10-500 ng/mL with extraction recovery ranging from 77.3 to 97.3% while all validation parameters met the acceptance criteria and proved the methods' reliability. The validated methods were successfully applied to plasma samples donated from cancer patient following treatment with temozolomide.

A new, sensitive and versatile assay for quantitative determination of α-fluoro-ß-alanine (AFBA) in human urine by using the reversed-phase ultrahigh performance-tandem mass spectrometry (rp-UHPLC-MS/MS) system.

A method for the quantitation of α-fluoro-ß-alanine (AFBA), the main metabolite of capecitabine (Cape) and 5-fluoruracil (5-FU), is described. Among antineoplastic drugs (ADs), 5-FU and Cape (the new oral prodrug) are the most commonly applied drugs in cancer therapy. The main objective of this study was to develop a reliable method that would be easy to run on a reversed-phase UHPLC system coupled to tandem mass spectrometry. AFBA was derivatized with Sanger's reagent to ensure complete yield of a stable 2,4 dinitrophenil-α-fluoro-ß-alanine derivative. This method was based on the use of a mixed-mode anion exchange solid phase extraction enabling urinary extracts to be clear of endogenous interferences affecting quantitative results. The assay was validated in human urine according to FDA criteria with the use of a labeled internal standard (ß-alanine-d4) to minimize experimental error. Good accuracy and precision were demonstrated by determining spiked urine QC samples in four consecutive days. The recovery of AFBA was between 70.0 and 82.6%, with a matrix effect that was 12.8%-18.5%. The lower limit of quantitation (LOQ) was 0.5 ng/mL with a coefficient of variation of 5.3%. This assay was successfully applied to determine the levels of this metabolite in a large number of urine samples taken from personnel who were occupationally exposed to ADs.

Determination of four Alternaria alternata mycotoxins by QuEChERS approach coupled with liquid chromatography-tandem mass spectrometry in tomato-based and fruit-based products.

A liquid chromatography-tandem mass spectrometry method for the determination of four Alternaria toxins, i.e. alternariol, alternariol monomethyl ether, tentoxin, and tenuazonic acid in tomato-based and fruit-based products was developed using a QuEChERs approach for the extraction of the mycotoxins. To optimise the QuEChERs extraction, several parameters were tested: types of QuEChERs pouches, sample weights, quantities of added water, use of dispersive SPE as a purification step, types of solvent and conditions of shaking. The method showed good linearity (R2 > 0.997) and precision (RSD% < 10) for all analytes. Tenuazonic acid showed very good recovery (98.8%-108.9%) for tomato-based products, as well as for fruit-based products. The method was successfully applied to 57 samples collected from the Italian market. Tenuazonic acid was found in appreciable concentrations in some products. The highest value was found in a tomato sauce sample (814 µg/kg).

Liquid chromatography-tandem mass spectrometric assay for the quantitation of the novel radiation protective agent and radiation mitigator JP4-039 in murine plasma.

JP4-039 radio-protects prior to, and radio-mitigates after ionizing radiation by neutralizing reactive oxygen species. We developed and validated an LC-MS/MS assay for the quantitation of JP4-039 in murine plasma. Methanol protein precipitation of 50µL plasma was followed by isocratic reverse phase chromatography for a 6min run time, and electrospray positive mode ionization mass spectrometric detection. The plasma assay was linear from 1 to 1000ng/mL with appropriate accuracy (97.1-107.6%) and precision (3.7-12.5%CV), and fulfilled FDA guidance criteria. Recovery was 77.2-136.1% with moderate ionization enhancement (10.9-39.5%). Plasma freeze-thaw stability (98.8-104.2%), stability for 13.5 months at -80°C (93.1-105.6%), and stability for 4h at room temperature (94.2-97.6%) were all acceptable. Limited cross-validation to tissue homogenates suggested that these could also be analyzed for JP4-039 accurately. This assay has been directly applied to determine the pharmacokinetics of JP4-039 in C57BL/6 male mice after IV administration of 20mg/kg JP4-039 and will be extended to other studies of this agent.

Development and validation of a bioanalytical method for quantification of LNA-i-miR-221, a 13-mer oligonucleotide, in rat plasma using LC-MS/MS.

LNA-i-miR-221, a 13-mer oligonucleotide, is a new miR-221 inhibitor that could be used as a novel drug for multiple myeloma. Herein, an ion-pair reversed phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of LNA-i-miR-221 in rat plasma. Plasma samples were prepared with an initial phenol/chloroform/isoamyl alcohol liquid-liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase at a flow rate of 0.4 mL/min. Under these conditions LNA-i-miR-221 and the analogue internal standard are co-eluted at 1.2 min. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The assay showed a good linearity within the calibration range 10-10000 ng/mL. The precision, accuracy, and recovery values were found to be <15% (<20% at LLOQ), 100 ±â€¯15%, and 97.6-103.7%, respectively. This method was successfully applied to measure the concentrations of LNA-i-miR-221 in plasma samples following the intravenous administration during a 4-week toxicity study in rats.

Stability assessment of antibody-drug conjugate Trastuzumab emtansine in comparison to parent monoclonal antibody using orthogonal testing protocol.

Antibody-drug conjugates (ADC) represent an emerging, novel class of biopharmaceuticals. The heterogeneity originating from the sophisticated structure requires orthogonal analytical techniques for quality and stability assessment of ADC to ensure safety and efficacy. In this study, the stability of Trastuzumab (recombinant humanized IgG1 mAb, targeting HER2 receptor) and its ADC with DM1 (anti-tubulin anticancer drug), Trastuzumab emtansine (T-DM1) were studied. SE-HPLC was used to monitor formation of aggregates and/or fragments of the monoclonal antibodies (mAb). Correlation with the results of reducing and non-reducing sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS) were performed to interpret the obtained results. RP-HPLC was used for assessment of the stability of DM1 in ADC while spectrophotometry was employed to determine drug antibody ratio (DAR) . The studied drugs were subjected to several stress conditions including pH, temperature, mechanical agitation and repeated freeze and thaw to generate possible degradation products and ensure suitability of the assay protocol. The degradation pattern and extent were demonstrated under the indicated stress conditions. The correlation between the results of SE-HPLC and those of SDS-PAGE and DLS ensured the validity of the orthogonal assay protocol and indicated aggregates that were not detected using SE-HPLC. Results showed clearly that T-DM1 is relatively less stable than its parent mAb. This was attributed to the presence of the drug-linker part that is attached to the mAb. RP-HPLC showed that the cytotoxic drug moiety is liable for degradation under the studied conditions resulting in alteration of DAR as well as formation of degradation products. This confirmed the need for more robust coupling chemistries for production of safe and effective ADC and highlighted the importance of orthogonal testing protocols for quality assessment. The assay protocol should be applicable for quality and stability assessment of various ADC.

Development & validation of reversed phase HPLC method for quantification of water insoluble API.

In the present work a specific, accurate, precise, and reproducible UV-HPLC method was developed and validated for the analysis of Aceclofenac. This method involved elution of Aceclofenac in a mobile phase which is composed of buffer pH 6.8 (i.e. using 0.01N KH2PO4) and HPLC grade Acetonitrile (60:40). Separation of the analyte was achieved using HPLC isocratic pump attached to the UV-VIS detectorC18, guard column and C18 column. The injection volume was 20µL, detected at 274 nm; flow rate: 1mL/min. Standard calibration curve was measured and found linear from 0.1 to 40µg/ml. The validation parameters were measured according to FDA guidelines and successful results were obtained. The presented analytical method could be employed for pharmacokinetic studies.

Evaluation of dynamics of derivatization and development of RP-HPLC method for the determination of amikacin sulphate.

The absence of chromophore and/or conjugated system, prerequisite for UV and florescent light detection, or absorbance at very low wavelength necessitates the development of simple and reliable methods for the determination of amikacin sulphate. Therefore, the present study describes for the first time dynamics of the drug derivatization using ninhydrin reagent and development and validation of a simple RP-HPLC method, using diode array detector (DAD). The variables such as heating time, heating type, drug-reagent ratio, reagent composition and storage temperature of the derivative were optimized. The analyte and aqueous ninhydrin solution upon heating for 2.00-5.00 min produced the colored drug-derivative which was stable for one month at refrigeration. The derivatized drug (20.00µL) was eluted through a column - Eclipse DB-C18 (5.00 µm, 4.60×150.00 mm), maintained at 25°C- using isocratic mobile phase comprising water and acetonitrile (70:30, v/v) at a flow rate of 1.00 mL/min, and detected at 400 nm. The method was found to be reliable (98.08-100.72% recovery), repeatable (98.02-100.72% intraday accuracy) and reproducible (98.47-101.27% inter day accuracy) with relative standard deviation less than 5%. The results of the present study indicate that the method is easy to perform, specific and sensitive, and suitable to be used for the determination of amikacin sulphate in bulk and pharmaceutical preparations using less expensive/laborious derivatization.

Identification and Quantification of Cyclic Di-Guanosine Monophosphate and Its Linear Metabolites by Reversed-Phase LC-MS/MS.

Cyclic dinucleotides such as bis-(3',5')-cyclic dimeric guanosine monophosphate (3',3'-c-di-GMP) represent an important class of second messengers in bacteria and are involved in numerous (patho)physiological settings. Here, we describe a sensitive and specific quantification method for 3',3'-c-di-GMP by HPLC-coupled tandem mass spectrometry (LC-MS/MS). Additionally, linear 3',3'-c-di-GMP metabolites, i.e., 5'-phosphoguanylyl-3',5'-guanosine (pGpG) and 5'-guanosine monophosphate (5'-GMP), as well as cyclic guanosine monophosphate (3',5'-cGMP) and 3',3' c-di-GMP analogues (2',3'-c-di-GMP and 2',2'-c-di-GMP) can be simultaneously determined by this method.