Optimized LC-MS/MS methods for quantifying antibody-drug conjugate payloads in cell culture media containing phenol red.

Aim: Phenol red is commonly used in cell culture media, but can be detrimental to bioanalysis of in vitro samples as it may impact instrument reliability. Many researchers do their final stage of culture in 'phenol red free' media, but in collaborative work this is not always feasible.Materials & methods: A comparison was made between typical extraction methods to reduce phenol red matrix interferences, including organic solvent precipitation and solid phase extraction.Results: The final method was demonstrated to be precise and accurate for the measurement of a target analyte by LC-MS/MS, and was applied to an in vitro ADC deconjugation study.Conclusion: This method allows for for continued bioanalytical support of in vitro models used in drug development.

[Box: see text].

Multifunctional Janus Membrane for Diabetic Wound Healing and Intelligent Monitoring.

The complex microenvironment of diabetic wounds often hinders the healing process, ultimately leading to the formation of diabetic foot ulcers and even death. Dual monitoring and treatment of wounds can significantly reduce the incidence of such cases. Herein, a multifunctional Janus membrane (3D chitosan sponge-ZE/polycaprolactone nanofibers-ZP) was developed by incorporating the zinc metal-organic framework, europium metal-organic framework, and phenol red into nanofibers for diabetic wound monitoring and treatment. The directional water transport capacity of the resulting Janus membrane allows for unidirectional and irreversible drainage of wound exudate, and the multifunctional Janus membrane creates up to a 99% antibacterial environment, both of which can treat wounds. Moreover, the pH (5-8) and H2O2 (0.00-0.80 µM) levels of the wound can be monitored using the color-changing property of phenol red and the fluorescence characteristic of Eu-MOF on the obtained membrane, respectively. The healing stages of the wound can also be monitored by analyzing the RGB values of the targeted membrane images. This design can more accurately reflect the wound state and treat the wound to reduce bacterial infection and accelerate wound healing, which has been demonstrated in in vivo experiments. The results provide an important basis for early intervention in diabetic patients.

Plasma catalysis. A study of the influence of oxysulfur, SOx• species through the degradation of sulfonated dyes by non-thermal plasma.

This work studied the degradation reaction of sulfonated dyes, indigo carmine, phenol red, and their mixtures by non-thermal plasma (NTP). Interestingly, the degradation rate constant showed a faster process and lower activation energy (Ea) for the dye mixtures than for the degradation reaction of the individual dyes. This unexpected result opened up new opportunities for understanding plasma chemistry and the interaction between reactive species formed by the plasma and the target molecule. As no catalyst or chemical additive was added to the reactor, the decrease in Ea came from a self-synergistic effect (SSE), through the dye molecules fragmentation, which resulted in plasma catalysis. The hypothesis proposed in this work is that oxysulfur (SOx) species are formed by the desulfonation reaction of dyes. The sulfonic groups (SO3) present in the chemical structures of dyes can function as precursors for forming several SOx•- species. Studies based on oxygenated sulfonated species such as SO3•-, SO4•- and SO5•- have been widely applied in advanced oxidative and reductive processes due to their satisfactory efficiency and low cost. Among them, SO4•- is the key reactive species with the best performance in the degradation of pollutants due to its high oxidation potential (E° = 2.60 V). In addition, it is an alternative source of HO• in aqueous media, improving the oxidation reaction. In order to elucidate the SSE, the kinetic process was followed by UV-Vis analysis, and the reactive species, such as alkyl, hydroxyl, and oxy-sulfur radicals were identified by Electron Paramagnetic Resonance. The by-products of the NTP degradation reaction were analyzed by ultrafast liquid chromatography coupled with a mass spectrometer, and a fragmentation route was proposed.

Development of a RP-HPLC method for simultaneous determination of atenolol, metoprolol tartrate and phenol red for in-situ rat intestinal perfusion studies.

Single-pass intestinal perfusion (SPIP) method is a widely used experimental model to determine the intestinal permeability of drugs. These studies are performed in the presence of a reference standard (metoprolol, MT) and a zero permeability marker (phenol red, PR). Therefore, it is important to develop a validated method for simultaneous determination of the investigated compound along with MT and PR. The aim of this study was to develop a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV-detection for the simultaneous determination of atenolol (ATN), MT, and PR in the perfusion medium used in SPIP experiments. Separation of compounds were performed using an InertSustain C18 (250 × 4.6 mm, 5 µm) HPLC column at 35 °C. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 7.0, 12.5 mM) in gradient elution, and was delivered at a flow rate of 1 mL/min. The acetonitrile ratio of the mobile phase increased linearly from 10 to 35 % over 15 min. The injection volume was 20 µL, and ATN, MT and PR were detected at 224 nm. The retention times under optimum HPLC conditions were 5.028 min, 12.401 min, and 13.507 min for ATN, MT and PR, respectively. The developed RP-HPLC method was validated for selectivity, specificity, calibration curve and range, accuracy and precision, carry-over effect, stability, reinjection reproducibility, recovery and robustness. The method was linear for ATN (0.76-50 µg/mL), MT (1.14-50 µg/mL), and PR (0.47-20 µg/mL) with determination coefficients of 0.9999, 0.9994 and 0.9998, respectively. The results obtained for all validation parameters of the developed RP-HPLC method met the required limits of the ICH M10 Guideline.

A smartphone-assisted sensing hydrogels based on UCNPs@SiO2-phenol red nanoprobes for detecting the pH of aquatic products.

For some aquatic products, pH has been considered a useful index to reflect the changes in materials during the loss of freshness. Based on the inner filter effect (IFE) between deprotonated phenol red (PR) and upconversion nanoparticles (UCNPs), UCNPs coated with PR-doped SiO2 shell were embedded in agarose hydrogel to develop a smartphone-assisted method for pH sensing. With the enhancement of pH response using a phase transfer agent (i.e., tetra butyl ammonium hydroxide, TBAH), the proposed senor realized the colorimetric and fluorescence detection of pH in the range of pH 6.6-8 and pH 6-8, respectively. The sensor also showed satisfied reversibility when switched between pH 6 and 8 for at least 5 cycles. Moreover, this sensor displayed great sensitivity, stability, and portability in analyzing actual fish, shrimp, and shellfish samples, providing a new sight for evaluating the freshness of aquatic products.

Development of a pH-responsive intelligent label using low molecular weight chitosan grafted with phenol red for food packaging applications.

In this study, we successfully developed a screen-printed pH-responsive intelligent label using low molecular weight chitosan grafted with phenol red (LCPR) as a colorant for screen printing ink. The LCPR was synthesized via a Mannich reaction, and its successful grafting was confirmed through FT-IR, UV-vis, and NMR spectroscopy. The LCPR exhibited lower crystallinity and thermal stability compared to low molecular weight chitosan (LC) and demonstrated zwitterionic behavior. To create intelligent labels, the LCPR-based ink was efficiently printed on cotton substrates with high resolution. The label exhibited remarkable sensitivity to buffer pH solutions and ammonia gas, leading to distinctive color changes from orange to red to purple. Additionally, the label showed excellent reversibility, storage stability, and leaching resistance to different food simulant solutions. The label was utilized to monitor shrimp freshness, successfully detecting a noticeable color shift upon spoilage. These findings highlight the significant potential of the LCPR-based label as an intelligent food packaging solution, offering pH-responsiveness and color stability for qualitative freshness detection of protein-rich food.

An Extensive Study of Phenol Red Thread as a Novel Non-Invasive Tear Sampling Technique for Proteomics Studies: Comparison with Two Commonly Used Methods.

Tear samples are considered in recent publications as easily, noninvasively collectible information sources for precision medicine. Their complex composition may aid the identification of biomarkers and the monitoring of the effectiveness of treatments for the eye and systemic diseases. Sample collection and processing are key steps in any analytical method, especially if subtle personal differences need to be detected. In this work, we evaluate the usability of a novel sample collection technique for human tear samples using phenol red threads (cotton thread treated with the pH indicator phenol red), which are efficiently used to measure tear volume in clinical diagnosis. The low invasiveness and low discomfort to the patients have already been demonstrated, but their applicability for proteomic sample collection has not yet been compared to other methods. We have shown, using various statistical approaches, the qualitative and quantitative differences in proteomic samples collected with this novel and two traditional methods using either glass capillaries or Schirmer's paper strips. In all parameters studied, the phenol red threads proved to be equally or even more suitable than traditional methods. Based on detectability using different sampling methods, we have classified proteins in tear samples.

Appraisal of Chitosan-Gum Arabic-Coated Bipolymeric Nanocarriers for Efficient Dye Removal and Eradication of the Plant Pathogen Botrytis cinerea.

The treatment of textile wastewater comprising many dyes as contaminants endures an essential task for environmental remediation. In addition, combating antifungal multidrug resistance (MDR) is an intimidating task, specifically owing to the limited options of alternative drugs with multitarget drug mechanisms. Incorporating natural polymeric biomaterials for drug delivery provides desirable properties for drug molecules, effectively eradicating MDR fungal growth. The current study fabricated the bipolymeric drug delivery system using chitosan-gum arabic-coated liposome 5ID nanoparticles (CS-GA-5ID-LP-NPs). This study focused on improving the solubility and sustained release profile of 5I-1H-indole (5ID). These NPs were characterized and tested mechanically as a dye adsorbent as well as their antifungal potencies against the plant pathogen, Botrytis cinerea. CS-GA-5ID-LP-NPs showed 71.23% congo red dye removal compared to crystal violet and phenol red from water and effectively had an antifungal effect on B. cinerea at 25 µg/mL MIC concentrations. The mechanism of the inhibition of B. cinerea via CS-GA-5ID-LP-NPs was attributed to stabilized microtubule polymerization in silico and in vitro. This study opens a new avenue for designing polymeric NPs as adsorbents and antifungal agents for environmental and agriculture remediation.

Synthesis of chitosan-based polymeric dyes as colorimetric pH-sensing materials: Potential for food and biomedical applications.

pH-sensitive polymeric dyes were fabricated by grafting phenol red (PR) and rosolic acid (RA) onto chitosan (CS) by a facile method. Successful grafting was confirmed by 1H NMR, FT-IR, UV-vis, XRD, and elemental analysis. The polymeric dyes exhibited no cell toxicity. The colorimetric pH-sensing films were fabricated by blending the polymeric dyes with CS to establish their pH-dependent color properties. The film color changed in the pH range 4-10, which may indicate food spoilage or wound status. Covalently grafting of polymeric dyes in the films led to excellent color stability, leaching resistance, and reversibility. Hence, the synthesized polymeric dyes had potential as pH-indicative colorants for food and biomedical fields.

Colorimetric loop-mediated isothermal amplification (LAMP) for cost-effective and quantitative detection of SARS-CoV-2: the change in color in LAMP-based assays quantitatively correlates with viral copy number.

We demonstrate a loop-mediated isothermal amplification (LAMP) method to detect and amplify SARS-CoV-2 genetic sequences using a set of in-house designed initiators that target regions encoding the N protein. We were able to detect and amplify SARS-CoV-2 nucleic acids in the range of 62 to 2 × 105 DNA copies by this straightforward method. Using synthetic SARS-CoV-2 samples and RNA extracts from patients, we demonstrate that colorimetric LAMP is a quantitative method comparable in diagnostic performance to RT-qPCR (i.e., sensitivity of 92.85% and specificity of 81.25% in a set of 44 RNA extracts from patients analyzed in a hospital setting).

Synthesis and Evaluation of PEG-PR for Water Flux Correction in an In Situ Rat Perfusion Model.

Phenol red (PR) is a widely used marker for water flux correction in studies of in situ perfusion, in which intestinal absorption usually leads to the underestimation of results. In this paper, we propose a novel marker polyethylene glycol (PEG)-PR (i.e., PR modified by PEGylation) with less permeability and evaluate its application in an in situ perfusion model in rats. PEG-PR was synthesized by the chemical conjunction of polyethylene glycol-4k/5k (PEG-4k/5k) and PR. The synthesized PEG-PR was then characterized using 1H-NMR, 13C-NMR, ultraviolet (UV), X-ray diffraction (XRD), and differential scanning calorimetry (DSC) analyses. The low permeability of PEG-PR was assessed using everted gut sac (EGS) methods. The apparent permeability coefficients (Papp, 3-8 × 10-7 cm/s) of PEG4k/5k-PR exhibited a nearly 15-fold reduction compared to that of PR. The different concentrations of PEG4k/5k-PR did not contribute to the Papp value or cumulative permeable percentage (about 0.02-0.06%). Furthermore, the larger molecular weight due to PEGylation (PEG5k-PR) enhanced the nonabsorbable effect. To evaluate the potential application of the novel marker, atenolol, ketoprofen, and metoprolol, which represent various biopharmaceutics classification system (BCS) classes, were selected as model drugs for the recirculation perfusion method. The water flux corrected by PEG4k/5k-PR reflected the accuracy due to the nonabsorbable effect, while the effective intestinal membrane permeability (Peff) of atenolol corrected by PEG4k/5k-PR showed a statistically significant increase (p < 0.05) in different intestinal segments. In conclusion, PEG-PR is a promising marker for the permeability estimation when using the in situ perfusion model in rats.

Rapid diagnosis of Theileria annulata by colorimetric loop-mediated isothermal amplification (LAMP) assay.

Theileria annulata the causative agent of bovine tropical theileriosis (BTT) is globally distributed. Rapid and accurate detection of the parasite is essential for the implementation and evaluation of mass drug administration and planned vaccination programs for controlling BTT. Loop mediated isothermal amplification (LAMP) amplifies targeted nucleic acid with a high efficacy, sensitivity and rapidity under isothermal conditions. In the present study, the internal transcriber space (ITS) gene of T. annulata was targeted for the development of a LAMP assay using pH-sensitive dye (phenol red) for enhanced visual detection of amplification. This method employed a set of specially designed four primers that recognized six distinct regions on the targeted gene. No amplification was detected with the DNA of other tested haemoprotozoans. Positive LAMP products were identified by a colour change from pink to yellow, and then rechecked by specific ladder pattern upon agarose gel electrophoresis. LAMP was able to detect infection in 63 out of 85 animals compared with blood microscopy, simple PCR and nested PCR which detected infection in 40, 49 and 64 animals, respectively. No difference in detection was seen in the colorimetric assay and the classical UV based LAMP assay.

Comparative Evaluation and Quantitative Analysis of Loop-Mediated Isothermal Amplification Indicators.

Loop-mediated isothermal amplification (LAMP) as a diagnostic tool is rapidly gaining recognition and maturity. Among various advantages over traditional polymerase chain reaction, the ability to visually detect amplification by the incorporation of colorimetric indicators is one of its most unique features. There is an overwhelming variety of LAMP indicators in the literature, yet a comprehensive comparative study is lacking. This study evaluates the use of hydroxynaphthol blue, phenol red, calcein, leuco crystal violet, malachite green, and a fluorescent dye for visual detection. A method for objective quantitative analysis using ImageJ is described that is readily implemented in standard and microfluidic workflows. The work here also includes the largest inter-reader variability study involving 24 participants to evaluate these indicators. We found inaccuracies in visual assessment as bias and/or individual-based perception can exist, solidifying the need for objective analysis. There was not a "universal" indicator, although considerations in sample preparation, storage, and applicability are discussed in length.

Relieving Sore Throat Formula Exerts a Therapeutic Effect on Pharyngitis through Immunoregulation and NF-κB Pathway.

Relieving Sore Throat Formula (RSTF) is a formula approved by the China Food and Drug Administration and has been used for the treatment of pharyngitis in clinic for many years. However, the potential pharmacological mechanism still remains unknown. We combined multiple methods including bioinformatics data digging, network pharmacology analysis, and pathway analysis to predict the potential target of RSTF. We verified our in silico prediction results with an in vivo/vitro antibacterial effect test, mouse phagocytic index test, proliferation, transformation, and migration of mouse spleen lymphocytes. Alteration of NF-κB pathway was determined by Western blotting, immunofluorescence, and PCR. The in vivo experiments demonstrated that the RSTF could significantly relieve the symptoms of pharyngitis. A rat saliva secretion test showed that RSTF can effectively relieve the xerostomia symptom. A phenol red excretion test showed that RSTF has an eliminating phlegm effect. A hot plate method and granuloma experiment proved that RSTF also have analgesic and anti-inflammatory effects. In silico prediction demonstrates that 70 active compounds of RSTF were filtered out through ADME screening and 84 putative targets correlated with different diseases. Pathway enrichment analysis showed that the candidate targets were mostly related to the response to bacteria and immunity signalling pathways, which are known contributors to pharyngitis. Experimental results confirmed that RSTF exerted therapeutic effects on pharyngitis mainly by antibacterial effect and downregulation of NF-κB activities. It is demonstrated both in silico and in vivo/vitro that RSTF exerted therapeutic effects on pharyngitis mainly through an antibiotic effect and downregulation of NF-κB signalling pathway.

Reversible Nontoxic Thermochromic Microcapsules.

Thermochromic materials exhibit a color change in response to a change in temperature. Creating nontoxic microcapsules containing thermochromic materials for applications in ink and film materials is historically challenging. In this study, we develop a nontoxic chlorophenol red (CPR)-water thermochromic system and its microcapsules with silicone shells via a reaction between water and octadecyltrichlorosilane (OTS) at the interface of a w/o emulsion. The obtained microcapsules exhibit a clear color change with full reversibility and are successfully used as inks by screen printing and as additives in films. Nontoxicity of both microcapsules and films is demonstrated through cell cytotoxicity assays. These features make these novel materials applicable to the next generation of intelligent sensors, coating, and food packaging materials.

Hemocompatible hemoadsorbent for effective removal of protein-bound toxin in serum.

Resin hemoperfusion is a life-saving treatment for drug intoxication or hepatic failure of patients. However, current resin adsorbents exhibit a limited hemocompatibility or low adsorption efficiency, representing a major roadblock to successful clinical applications. In this work, we developed a hemocompatible and effective hemoadsorbent based on polystyrene resin (H103) microparticles encapsulated in anti-biofouling zwitterionic poly(carboxybetaine) (PCB) hydrogels. Apart from a strong mechanical stability, this PCB-based adsorbent (PCB-H103) exhibited excellent hemocompatibility (hemolysis ratio was ∼0.64%), which was attributed to the anti-biofouling property of PCB hydrogel. In addition, it can efficiently adsorb both small and middle molecular weight molecules in phosphate-buffered saline, and the efficiencies were significantly higher than poly(ethylene glycol) methacrylate-based and poly(2-hydroxyethyl methacrylate)-based adsorbent counterparts, indicating the favorable permeability of PCB hydrogel coating. More importantly, PCB-H103 could effectively remove protein-bound toxins including phenol red and bilirubin in bovine serum albumin solution or even in 100% fetal bovine serum (FBS). In 100% FBS, the adsorption capacity of PCB-H103 towards bilirubin was 8.3 times higher than that of pristine clinical-scale resin beads. Findings in this work may provide a new strategy for the development of modern resin hemoperfusion technology.

In vitro tissue culture model validation-the influence of tissue culture components on IPL energy output.

Intense pulsed light (IPL) has been used therapeutically in a number of clinical settings and has been shown to have a photobiomodulatory effect on connective tissue cells, such as those derived from skin and tendon. In vitro cell culture models are essential tools preclinically in investigating such treatment modalities, as they help in optimising parameters for successful treatment. However, as culture system components have been reported to absorb part of the irradiated energy, which in turn has a bearing on the amount of light reaching the cells, it is important to establish specific parameters for the particular in vitro model used. This study, therefore, investigates the effect of our tissue culture system components on the IPL energy delivered. Individual wells of multi-well plates were irradiated with IPL at different device settings and under variable culture conditions (e.g. in the absence or presence of cell culture media with or without the pH indicator dye, phenol red), and the energy lost through the culture system determined. Our data demonstrated that the IPL device delivered significantly lower outputs than those published, and energy absorption by the culture equipment would further reduce fluencies delivered to the cell monolayer. Furthermore, energy absorption by media containing phenol red was marginally greater than clear media and resulted in only a small increase in temperature, which would not be harmful to cells. The use of phenol red-containing media therefore is valid and physiologically relevant when examining light-culture system interactions.

Caution for the routine use of phenol red - It is more than just a pH indicator.

Phenol red (PR) is the standard pH indicator in various cell and tissue culture media, as it provides a quick check for the health of the culture. PR has also been used in multiple protocols to detect cellular hydrogen peroxide as well as peroxidase activity from human peroxidase enzymes. The majority of promyelocytic leukemia cell lines (e.g. HL-60 cells) express myeloperoxidase (MPO), which may react with PR, especially as the latter is present in cell culture media at sufficient concentrations (~15 µM) to partake in redox reactions. Moreover, phenolic molecules are often efficient donor substrates for peroxidase enzymes. In this study, we hypothesized that MPO metabolism of PR via MPO-expressing HL-60 cells could result in PR metabolite(s) that could modulate cell viability. We used purified human MPO for UV-visible spectrophotometry, electron paramagnetic resonance (EPR) and LC-MS analyses to investigate PR peroxidation. 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (monochloro-dimedone, MCD) was used to assess the effect of PR on MPO-catalyzed chlorination activity, and we assessed PR uptake by HL-60 cells using LC-MS analysis. Lastly, we investigated the impact of PR metabolism by intracellular MPO on cell viability (ATP, using CellTiter-Glo®), cytotoxicity (using trypan blue), and on reduced and oxidized glutathione (using GSH/GSSG-Glo™). Our results demonstrate that PR undergoes oxidative halogenation via MPO, resulting in its UV-vis spectral changes due to the formation of mono- and di-halogenated products. Moreover, a significant increase in MPO-catalyzed chlorination of MCD and an increase in glutathionyl radical detection (using EPR) were observed in the presence of PR. Our in-vitro studies revealed that PR is readily taken up by HL-60 cells and its metabolism by intracellular MPO leads to a significant decrease in cellular glutathione as well as a significant increase in glutathione disulphide formation. In spite of the latter, PR had no considerable effect on HL-60 cell viability. These results provide evidence that while no overt decrease in cell viability may be observed, PR does impart redox activity, which investigators should be wary of in experimental protocols.

Electroanalytical properties of chlorophenol red at disposable carbon electrodes: Implications for Escherichia coli detection.

The use of coliforms and Escherichia coli as indicator species for assessing the quality of water is well established and a large variety of methods based on ß-galactosidase (B-GAL) activity, inherent to the microbes within this classification, have arisen to enable their detection and enumeration. Chlorophenol red (CPR) is widely used as a chromogenic label, but its capacity for translation to electroanalytical devices has yet to be fully explored. The CPR moiety is capable of undergoing oxidation at carbon substrates (+0.7 V) giving rise to a variety of phenolic intermediates. Electrochemical, XPS and enzymatic techniques were employed to characterise the underpinning chemistry and the intermediate identified as a 1,2-quinone derivative in which the chlorine substituent is retained. The latter was found to accumulate at the electrode and, in contrast to the parent CPR, was found to be detected at a significantly less positive potential (+0.3 V). Bacterial hydrolysis of a CPR labelled substrate was demonstrated with the 1,2-quinone oxidation product found to accumulate at the electrode and detected using square wave voltammetry. Proof of concept for the efficacy of the alternative electrode pathway was established through the detection of E.coli after an incubation time of 2.5 h with no interference from the labelled substrates.

Extraction-free spectrophotometric assay of the antitussive drug pentoxyverine citrate using sulfonephthalein dyes.

Pentoxyverine citrate (PEN-citrate) is an antitussive (cough suppressant) drug used for cough associated with illnesses like common cold. In this work, PEN-citrate is quantified by applying a simple, direct and accurate spectrophotometric method in pure form, pharmaceutical formulation (Cabella®, 2.13 mg/mL) and human serum samples. The formation of a stable yellow ion-pair with sulfonephthalein dyes; bromocresol green (BCG), bromophenol blue (BPB), bromothymol blue (BTB), bromocresol purple (BCP), bromochlorophenol blue (BChPB) and bromoxylenol blue (BXB), in three nonpolar solvents (chloroform, dichloromethane, acetonitrile) is used as the basis for this method. This is the first assay method reported for the quantification of PEN-citrate using the sulfonephthaleins as coloring agents. Diverse parameters were investigated in order to optimize the calibration curve conditions. The strategy was validated with respect to linearity range, precision, accuracy, specificity, robustness and limits of detection (LOD) and quantification (LOQ). In addition, solvents of different polarities were utilized to investigate the color reaction, light absorption and to allow for increasing the method sensitivity. Beer's law is obeyed over a wide concentration range (up to 42.05 µg/mL in case of BTB method). LOD and LOQ values reached 0.22 and 0.72 µg/mL, respectively, upon using BChPB. The relative standard deviation (%RSD) was ≤1.91% while correlation coefficient values (r) were ≥ 0.9974. High molar absorptivity values and low values of Sandell's sensitivity were obtained indicating that the proposed methods are highly sensitive. The validated methods were applied to the analysis of PEN-citrate in the dosage form and human serum samples where the drug was successfully resolved from the pharmaceutical additives and serum components with recoveries ≥98.98%.