Polymicrobial biofilms resistant to antibiotic therapy are often associated with different severe human diseases, including skin and soft tissue injuries, local mucosal and dental infections, post-traumatic and chronic osteomyelitis, and implant-associated infections. A possible treatment option for these challenging infections may be the use of enzybiotics containing bacteriolytic enzymes (or lysins). However, elimination of polymicrobial infections requires an effective combination of enzymes to expand the narrow activity spectrum of individual lysins and to ensure successful treatment. We screened ten bacteriolytic enzymes for their antibacterial and biochemical properties, including effective doses, rate of action, spectrum of action, as well as effects of pH, temperature, and buffer systems on enzymes activity. The selected Gram-negative bacteria-targeting enzyme was further modified with an antimicrobial peptide to improve its stability. This engineered lysin GRC-ML07 was studied in combination with Gram-positive bacteria-targeting enzymes. Our results convincingly demonstrate that combinations of lysins with different substrate specificity and Gram specificity result in synergistic and additive effects against pathogenic bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacterales. Importantly, this was shown for both planktonic cells and biofilms, including polymicrobial consortia, as combinations significantly decreased bactericidal concentrations of enzymes against Gram-positive and Gram-negative bacteria and improved antibiofilm properties. This novel antibacterial enzybiotic composition was also safe in in vitro experiments.

Due to their availability and environmental friendliness, alginate polymers are widely used in pharmaceuticals and cosmetics. The most common type of alginate is derived from seaweed and is used to develop topical dosage forms, among other things. However, variability in the seaweed material can lead to instability in the physicochemical parameters. Biotechnologically produced alginate minimizes this drawback through controlled synthesis. However, unlike algal alginates, the safety profile of such polymers has not been well studied. When developing dosage forms intended for wound surfaces, safety is of primary importance. In this study, we developed enzybiotic compositions based on bacterial alginate as an excipient and a novel recombinant modified endolysin, LysSi3-LK, as an antibacterial agent, and assessed their antibacterial properties and safety profile. The study included an in vitro evaluation of the activity spectrum, as well as the cytotoxicity and biocompatibility, of gel and hydrogel compositions. It was demonstrated that bacterial alginate is acceptable for the encapsulation of endolysin. It exhibited medium cytotoxic effects on the HaCaT cells, which were significantly reduced by the LysSi3-LK addition. The migration of cells was diminished following exposure to the gel and hydrogel formulations. However, an improvement in biocompatibility was observed in the cell proliferation assay.

With the rise of antibiotic resistance, there is a need for innovative drugs with alternative mechanisms of action. Endolysins meet most of the requirements, but are limited for parenteral use due to their short blood circulation time. In this article, a number of modifications to the ML06-engineered, lysin-targeting Gram-negative bacteria are proposed to improve its pharmacokinetic parameters. Genetic modification with albumin-binding and dimerization domains ranging from 11–12 aa to 45 aa at both the C- and N-termini has resulted in six enzymes that do not exhibit critically reduced antibacterial properties in vitro, and in the case of the ABP1 modification, an improved antibacterial rate and spectra of enzymes. The ML06-ABP1, ML06-ABP2, and HDD-ML06 modifications also retained activity in blood serum and significantly increased serum stability. A pharmacokinetic study of the three modifications in mice showed that ML06-ABP2 and HDD-ML06 have a prolonged half-life compared to the ML06 half-life. In addition, the serum Cmax concentration for HDD-ML06 (22.2 μg/mL) was significantly increased compared to ML06 (Cmax < 5 μg/mL). Our results allow for a comparison of the different types of modifications that are useful in the development of parenteral antibacterials.

The development of new and effective antibacterials for pharmaceutical or cosmetic skin care that have a low potential for the emergence and expansion of bacterial resistance is of high demand in scientific and applied research. Great hopes are placed on alternative agents such as bactericidal peptidoglycan hydrolases, depolymerases, etc. Enzybiotic-based preparations are being studied for the treatment of various infections and, among others, can be used as topical formulations and dressings with protein-polysaccharide complexes. Here, we investigate the antibiofilm properties of a novel enzybiotic cocktail of phage endolysin LysSi3 and bacteriocin lysostaphin, formulated in the alginate gel matrix and its ability to control the opportunistic skin-colonizing bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae, as well as mixed-species biofilms. Our results propose that the application of SiL-gel affects different components of biofilm extracellular polymeric substances, disrupts the matrix, and eliminates the bacteria embedded in it. This composition is highly effective against biofilms composed of Gram-negative and Gram-positive species and does not possess significant cytotoxic effects. Our data form the basis for the development of antibacterial skin care products with a gentle but effective mode of action.

Among the non-traditional antibacterial agents in development, only a few targets critical Gram-negative bacteria such as carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii or cephalosporin-resistant Enterobacteriaceae. Endolysins and their genetically modified versions meet the World Health Organization criteria for innovation, have a novel mode of antibacterial action, no known bacterial cross-resistance, and are being intensively studied for application against Gram-negative pathogens. The study presents a multidisciplinary approach, including genetic engineering of LysECD7-SMAP and production of recombinant endolysin, its analysis by crystal structure solution following molecular dynamics simulations and evaluation of antibacterial properties. Two types of antimicrobial dosage forms were formulated, resulting in lyophilized powder for injection and hydroxyethylcellulose gel for topical administration. Their efficacy was estimated in the treatment of sepsis, and pneumonia models in BALB/c mice, diabetes-associated wound infection in the leptin receptor-deficient db/db mice and infected burn wounds in rats. In this work, we investigate the application strategies of the engineered endolysin LysECD7-SMAP and its dosage forms evaluated in preclinical studies. The catalytic domain of the enzyme shares the conserved structure of endopeptidases containing a putative antimicrobial peptide at the C-terminus of polypeptide chain. The activity of endolysins has been demonstrated against a range of pathogens, such as Klebsiella pneumoniae, A. baumannii, P. aeruginosa, Staphylococcus haemolyticus, Achromobacter spp, Burkholderia cepacia complex and Haemophylus influenzae, including those with multidrug resistance. The efficacy of candidate dosage forms has been confirmed in in vivo studies. Some aspects of the interaction of LysECD7-SMAP with cell wall molecular targets are also discussed. Our studies demonstrate the potential of LysECD7-SMAP therapeutics for the systemic or topical treatment of infectious diseases caused by susceptible Gram-negative bacterial species and are critical to proceed LysECD7-SMAP-based antimicrobials trials to advanced stages.

The potential of new genetically modified recombinant endolysins as antimicrobial agents against Gram-negative bacteria was investigated. A series of enzymes based on LysSi3 lysozyme-like muramidase was obtained by modifying its sequence with antimicrobial peptides of different families, and recombinant expression in E. coli was demonstrated. Modification of LysSi3 resulted in increased bacteriolytic activity against the model isolate of A. baumannii and a higher kinetics rate compared to the native enzyme. The cytotoxic properties of the newly engineered lysins were investigated with the HEK293 and HaCaT cell lines, and it was shown that modification of LysSi3 with antimicrobial peptides does not significantly increase the toxic properties in vitro.

In recent years modified bacteriophage lysins have widely been investigated for the purposes of development of antibacterial therapy. Thus, effective and precise methods for the quantitative analysis of these enzymes are in high demand. The enzyme-linked immunosorbent assay (ELISA) method has been developed for the detection of recombinant modified endolysin LysAm24-SMAP in biological samples. The optimal parameters for protein detection were determined, in particular, the influence of salt and the composition of the buffer system for preparation of the samples was studied. The applicability of the immunodetection system of the genetically engineered endolysin LysAm24-SMAP in various biological samples with enzyme concentrations from 0.4 ng/mL was demonstrated. In addition, the influence of matrix effects in samples of animal organs and tissue homogenates and producer strain lysates and their individual components during the analysis was assessed and it was shown that 0.65 M NaCl addition in the ELISA buffer is crucial for achieving correct results and reduces nonspecific interactions in the case of LysAm24-SMAP. The effectiveness of the developed system in the immunochemical control of the bacteriolytic enzyme was confirmed.

Pharmacokinetics and safety studies of innovative drugs is an essential part of drug development process. Previously we have developed a novel drug for intravenous administration (lyophilizate) containing modified endolysin LysECD7-SMAP that showed notable antibacterial effect in different animal models of systemic infections. Here we present data on pharmacokinetics of endolysin in mice after single and multiple injections. Time-concentration curves were obtained, and pharmacokinetic parameters for preparation (C0, kel t1/2, AUC0-∞, MRT, ClT, Vss) were calculated. It was shown that although endolysin is rather short-lived in blood serum (t1/2 = 12.5 min), the therapeutic concentrations of LysECD7-SMAP (in degraded and non-degraded form) were detected for 60 minutes after injection that is sufficient for antibacterial effect. Based on the obtained data, it was proposed that endolysin distributes presumably in murine blood, degrades in blood and liver, and is eliminated via glomerular filtration. Safety profile of the preparation relating to general toxicity, immunotoxicity and allergenicity was assessed in rodents. It was demonstrated that LysECD7-SMAP in potential therapeutic (12.5 mg/kg), 10-fold (125 mg/kg) and 40-fold (500 mg/kg) doses showed no signs of intoxication and significant abnormalities after single and repeated i.v. administrations, preparation was non-immunogenic and induced minor and reversible allergic reaction in animals.

INTRODUCTION. The safe use of lily-of-the-valley medicinal products requires adequately controlling the content of cardiac glycosides. The State Pharmacopoeia of the Russian Federation stipulates that the content of cardiac glycosides in lily-of-the-valley medicinal products should be quantified using bioassays and spectrophotometry. However, the determination of cardiac glycosides needs more accurate and selective physicochemical methods, such as high-performance liquid chromatography (HPLC).AIM. This study aimed to develop and validate an HPLC analytical procedure for the quantitative determination of cardiac glycosides (convallatoxin) in lily-of-the-valley herbal medicinal products.MATERIALS AND METHODS. The study examined the Lily-of-the-Valley Tincture, Zelenin Drops, Valocormid, Carniland®, and Lily-of-the-Valley Extract Reference Standard samples. The content of cardiac glycosides was determined by comparison with a reference standard for convallatoxin by HPLC and spectrophotometry. The samples were prepared as outlined in Monograph 3.4.0003.18 Lily-of-the-Valley Herb Tincture of the State Pharmacopoeia of the Russian Federation. Mixed cardiac glycosides were separated on a Luna 5 µm C18(2) column by gradient elution with 0.1% orthophosphoric acid in water and acetonitrile. The analysis was performed using an autoinjector with sample cooling to 5 °C.RESULTS. The developed analytical procedure met the acceptance criteria for specificity, intermediate precision, linearity (correlation coefficient of 0.99985), and repeatability (relative standard deviation of convallatoxin measurements of 1.61%). The analytical procedure is suitable for the quantitative determination of convallatoxin in lily-of-the-valley herbal medicinal products because it produces reliable and repeatable results.CONCLUSIONS. The authors developed a highly sensitive and selective HPLC analytical procedure for the quantitative determination of convallatoxin in lily-of-the-valley herbal medicinal products. The content of convallatoxin ranged from 0.012 to 0.018 mg/mL in the liquid active substance Lily-of-the-Valley Tincture, from 0.004 to 0.013 mg/mL in the medicinal product Zelenin Drops, and from 0.005 to 0.007 mg/mL in the medicinal products Carniland® and Valocormid. For the Lily-of-the-Valley Extract Reference Standard, the content of convallatoxin amounted to 0.029 mg/mL.

The ability of most opportunistic bacteria to form biofilms, coupled with antimicrobial resistance, hinder the efforts to control widespread infections, resulting in high risks of negative outcomes and economic costs. Endolysins are promising compounds that efficiently combat bacteria, including multidrug-resistant strains and biofilms, without a low probability of subsequent emergence of stable endolysin-resistant phenotypes. However, the details of antibiofilm effects of these enzymes are poorly understood. To elucidate the interactions of bacteriophage endolysins LysAm24, LysAp22, LysECD7, and LysSi3 with bacterial films formed by Gram-negative species, we estimated their composition and assessed the endolysins’ effects on the most abundant exopolymers in vitro. The obtained data suggests a pronounced efficiency of these lysins against biofilms with high (Klebsiella pneumoniae) and low (Acinetobacter baumannii) matrix contents, or dual-species biofilms, resulting in at least a twofold loss of the biomass. These peptidoglycan hydrolases interacted diversely with protective compounds of biofilms such as extracellular DNA and polyanionic carbohydrates, indicating a spectrum of biofilm-disrupting effects for bacteriolytic phage enzymes. Specifically, we detected disruption of acid exopolysaccharides by LysAp22, strong DNA-binding capacity of LysAm24, both of these interactions for LysECD7, and neither of them for LysSi3.

Background/Objectives: Frequently, healthcare-associated and community-acquired skin and soft tissue infections develop in the context of the formation of persistent, multidrug-resistant polymicrobial consortia involving both Gram-negative and Gram-positive pathogens. Non-traditional antibacterials, in particular phage- and bacteria-derived lytic enzymes, are currently the focus of attention. However, in practice their action is not sufficient for a significant effect when used as monotherapy. Here, we propose an enzyme combination approach to obtain an effective antimicrobial composition and to enhance the efficacy of enzyme-based therapy. Methods: The enzymes individual antibacterial properties and the effects of the compositions were assessed in vitro. In vivo evaluation of a combined enzymes preparation was assessed in murine wound infection against polymicrobial biofilm containing Pseudomonas aeruginosa and Staphylococcus aureus. Results: Significant synergy or additive effects of lysins combinations were shown for S. aureus strains, while indifferent or additive effect – for Gram-negative species. Treatment of mixed biofilm-colonized wounds in an animal model showed that the combination halted the chronic process and systematization of infection and promoted accelerated tissue regeneration at the wound site. Conclusions: The combinations of peptidoglycan hydrolases with different substrate specificities can significantly increase the efficacy of the composition against Gram-positive and Gram-negative bacteria resulting in improved in vitro and in vivo antibacterial properties and has a potential to treat polymicrobial infections.

Antibacterial therapy with phage-encoded endolysins or their modified derivatives with improved antibacterial, biochemical and pharmacokinetic properties is one of the most promising strategies that can supply existing antibacterial drugs array. Gram-negative bacteria-induced infections treatment is especially challenging because of rapidly spreading bacterial resistance. We have developed modified endolysin LysECD7-SMAP with a significant antibacterial activity and broad spectra of action against gram-negative bacteria. Endolysin was formulated in a bactericidal gel for topical application with pronounced effectivity in local animal infectious models. Here we present preclinical safety studies and pharmacokinetics of LysECD7-SMAP-based gel. We have detected LysECD7-SMAP in the skin and underlying muscle at therapeutic concentrations when the gel is applied topically to intact or injured skin. Moreover, the protein does not enter the bloodstream, and has no systemic bioavailability, assuming no systemic adverse effects. In studies of general toxicology, local tolerance, and immunotoxicology it was approved that LysECD7-SMAP gel local application results in the absence of toxic effects after single and multiple administration. Thus, LysECD7-SMAP-containing gel has appropriate pharmacokinetics and can be considered as safe that supports the initiation of the phase I clinical trials of novel antibacterial drug intending to treat acute wound infections caused by resistant gram-negative bacteria.

An Erratum to this paper has been published: https://doi.org/10.1134/S0003683824030013

When used to quantify alkaloids in thermopsis dry extract, acidimetric titration has several limitations, including insufficient specificity of the method and arbitrary selection of a titration endpoint. Different parts of the Thermopsis lanceolata plant produce alkaloids in different proportions: the herb is rich in thermopsine, whereas the seeds are rich in cytisine. Since thermopsine and cytisine have different pharmacological effects, it is important to identify and quantify individual alkaloids in thermopsis dry extract. The aim of the study was to develop and validate an analytical procedure for identifying and quantifying total alkaloids in thermopsis dry extract by high-performance thin-layer chromatography (HPTLC) with densitometric detection. Materials and methods . The study included samples from two pilot-scale batches of thermopsis dry extract and reference standards for cytisine and thermopsine. The authors used Merck HPTLС Silica Gel 60 F 254 20×10 cm plates for chromato-graphic separation and analysed the results with a CAMAG® TLC Scanner 4 densitometer and the winCATS software. Results. The authors identified thermopsis dry extract using HPTLC separation followed by spectrodensitometry. The alkaloid-specific solution applied to chromatography plates helped to identify the three most abundant and four minor alkaloids of thermopsis. The most abundant alkaloids were thermopsine, cytisine, and an unidentified alkaloid with a retention factor of approximately 0.2. These three alkaloids accounted for almost 80% of the total alkaloid content of the dry extract. The authors quantified cytisine, thermopsine, and total alkaloids expressed as thermopsine. Conclusions. The authors developed and validated an analytical procedure for identifying and quantifying total alkaloids in thermopsis dry extract. This procedure offers the possibility of reducing the analysis time from 4–5 hours to 2–2.5 hours.

The demand for new treatment and prevention strategies of bacterial infections is increasing with the worldwide spread of acquired antimicrobial resistance among the microorganisms. This is especially important for healthcare-associated infections, exposed to a significant selective pressure of a closed system of in-patients organizations and pathogens' propensity to the resistance factors transmission. In this work, we assessed the in vitro potential of a new therapy approach against Gram-negative species based on combination of conventional antibiotics and a modified bacteriophage-derived endolysin LysECD7-SMAP, belonging to the class of enzybiotics. We have shown that such combinations can not only manifest a synergistic effect, but also restore the sensitivity of the microorganism to the antibiotic. The mechanism of the endolysins antimicrobial action suggests absence or low risks of development of bacterial resistance, which means that such a therapeutic regimen could be safe for course application. This study can serve as a basis for the development of new treatment regimens for patients with severe, recurrent or relapse infections caused by drug-resistant pathogens.

Scientific relevance. The State Pharmacopoeia of the Russian Federation, edition XIV, requires the identification of aniseed and fennel fruits by the same thin-layer chromatography (TLC) procedure using Sudan III and menthol. These markers are neither specific nor related to the therapeutic effects of these herbal drug products. The visual interpretation of chromatograms is complicated because of the low intensity of adsorption zones. Moreover, the corresponding compendial monographs do not characterise the adsorption zones specific to each of the plants. The most abundant component in aniseed and fennel essential oils, trans-anethole, would make a better reference standard from a methodological point of view.Aim. This study aimed to optimise the TLC procedure for essential oil determination in herbal drugs and herbal medicinal products of aniseed and fennel fruits and subsequently recommend it for inclusion in the relevant compendial monographs.Materials and methods. The study examined samples from several batches of herbal drugs, including aniseed and common fennel fruits, sourced from Russian manufacturers. The reference standards comprised commercial reagents, including trans-anethole, anise oil, and linalool, as well as fresh essential oils that had been steam distilled from the test samples in a Clevenger apparatus. The study was conducted by TLC. Sample preparation involved using Merck aluminium TLC plates, an IKA KS-501 digital orbital shaker, and a CAMAG Linomat 5 semi-automatic sample application system. The authors heated the plates in either a Binder ED53 drying oven or a CAMAG TLC plate heater. For visualisation, the authors selected a CAMAG TLC VISUALIZER 2 UV imaging and documentation system.Results. The informational and experimental research showed the feasibility of using trans-anethole as an identification reference standard for common anise and fennel fruits. The authors selected the optimal solvent for extracting active substances from the test products (hexane) and a detection reagent for improving visual evaluation of the chromatograms (1% sulfovanillin). The authors established characteristic adsorption zones for differentiating between aniseed and fennel fruits.Conclusions. The optimised procedure identifies the main components in the essential oils of aniseed and fennel fruits with a specific reference standard. This procedure may be recommended for inclusion in draft monographs for the corresponding herbal medicinal products.

Ascorbic acid (vitamin C) is involved in many vital biochemical processes in the human body. Rose hips are a natural source of ascorbic acid. Rose hips are used in decoctions and many vitamin herbal teas. Russian compendial methods for the quantitative determination of ascorbic acid have certain limitations precluding its objective measurement in herbal drugs, herbal drug preparations, and herbal medicinal products. The aim of the study was to develop an analytical procedure for the quantitative determination of ascorbic acid in herbal drugs, herbal medicinal preparations, and herbal medicinal products using high performance liquid chromatography (HPLC). Materials and methods . The study involved two herbal medicinal products, Rose Hips (rose hips) and Vitamin Herbal Tea 2 (rose hips and rowan fruits), and reference standards for ascorbic acid, rutoside, quercetin, gallic acid, caffeic acid, chlorogenic acid, luteolin-7-glucoside, citric acid, DL-malic acid, thiamine, and pyridoxine hydrochloride. The authors used a 1260 Infinity II chromatograph with a diode array detector by Agilent and an Inertsil ODS-3 chromatographic column (250 mm × 4.6 mm, 5 µm) by GL Sciences. The HPLC system was operated in gradient elution mode, and the detector was set at 244 nm. Results. Ascorbic acid content in herbal drugs, herbal medicinal preparations, and herbal medicinal products can be determined by HPLC. Adequate sample preparation and chromatography conditions allow for inhibiting ascorbic acid oxidation in aqueous solutions for a period sufficient to complete testing (not less than 8 h). Conclusions. The authors developed a highly sensitive and selective HPLC procedure for the quantitative determination of ascorbic acid intended for the standardisation of herbal drugs, herbal medicinal preparations, and herbal medicinal products. The procedure is worthy of inclusion in the Rose Hips and Vitamin Herbal Tea 2 monographs of the State Pharmacopoeia of the Russian Federation. Switching to HPLC should not require lowering the limit for ascorbic acid (not less than 0.2%) established in the Rose Hips and Vitamin Herbal Tea 2 monographs for the titration method. The ascorbic acid content was below this limit in the vast majority of study samples. However, this discrepancy may be explained by the presence of dog rose (Rosa canina) species low in vitamin C, which should normally be used only as choleretics.

Currently, there is an increase in pre- and post-approval testing of medicinal products containing diosmin and hence a need to unify approaches to standardisation of this group of pharmaceuticals. Moreover, the State Pharmacopoeia of the Russian Federation lacks a monograph for these products.The aim of the study was to determine an approach to standardisation of medicinal products containing diosmin.Materials and methods: the study analysed scientific publications, as well as monographs of leading foreign pharmacopoeias. Experimental work was carried out using samples of diosmin-containing pharmaceuticals in the form of 500 and 1000 mg film-coated tablets produced by Russian and foreign manufacturers. The study involved high performance liquid chromatography with UV detection using an Agilent 1260 Infinity II liquid chromatography system with a diode array detector. The following reference standards were used: a diosmin RS, USP grade; a hesperidin CRS, Ph. Eur. Grade; and a diosmin CRS for testing chromatography system suitability for identification of impurities A, B, C, D, E, and F, Ph. Eur. grade.Results: the authors reviewed quality requirements for pharmaceutical products containing diosmin and analysed experimental data obtained during pre- and post-approval testing of Russian and foreign medicines. The comparison of regulatory documents for registered diosmin-containing medicinal products showed a difference in approaches to assessing the contents of related substances and active pharmaceutical ingredients. Having analysed the literature, experimental data and regulatory requirements for standardisation of diosmin-containing pharmaceuticals, the authors recommended an approach to standardisation. According to the approach, concomitant flavonoids (hesperidin, isorchoifolin, linarin, and diosmetin) contributing to the pharmacological activity of a medicinal product are specified as part of Assay, and process-related by-products (impurities A and D) are specified and evaluated as part of Related substances tests.Conclusion: the authors propose to evaluate the contents of concomitant flavonoids (hesperidin, isorchoifolin, linarin, diosmetin) under Assay and to specify impurities A and D, as well as single unidentified impurities and total amount of impurities under Related substances.

Arbutin is the main active ingredient in many herbal medicinal products that have diuretic, antimicrobial, bactericidal, and antioxidant effects. Many of these products are mixtures of different herbal substances. Therefore, the approaches to quality control of HMPs can vary significantly. The aim of the study was to compare arbutin assay procedures used in quality control of arbutin-containing products. Materials and methods. Samples of the following HMPs were used in the study: monocomponent Bearberry Leaf and multicomponent Brusniver® (herbal mixture, powder). The test methods used were titrimetry, ultraviolet-visible spectrophotometry, and high-performance liquid chromatography (HPLC). Results. The authors compared five arbutin assay procedures described in the monographs and product files for arbutin-containing HMPs. Conclusions. It has been established that the analysed procedures cannot be used interchangeably as equivalent test methods; the limits for arbutin have to be established for each specific procedure. Iodometric titration is the most labour- and time-consuming method, and the determined titration endpoint is a subjective assessment. Spectrophotometric methods do not require the use of an arbutin reference standard, but they can give overestimated results as compared to the HPLC and titrimetry methods. HPLC methods are more selective, but they require the use of reference standards. The recommended test methods for HMP quality control are HPLC and visible spectrophotometry; the titrimetric method is recommended for replacement.