High-performance Liquid Chromatography Technique Research Articles

Composition and α-tocopherol content of Njeguski-type cheese made from cow, ewe and goat milk

The determination of basic composition and α-tocopherol content during ripening of Njeguski-type cheeses produced from cow, ewe and goat milk was studied. The α-tocopherol content was determined by high performance liquid chromatography (HPLC) technique with fluorescent detection. The results showed that although with the same farm practice and production technology applied, cheese composition of three types of milk differed significantly. Therefore, to standardise Njeguski cheese composition, production technology should be modified for each milk type. Additionally, the content of α-tocopherol in goat cheeses was significantly higher (P < 0.05) than the α-tocopherol content in cow and ewe cheeses: 1573 μg 100 g−1, 1039 μg 100 g−1, and 1152 μg 100 g−1, respectively, at the end of ripening.

Open-Label Study to Evaluate the Efficacy of a Topical Anhydrous Formulation with 15% Pure Ascorbic Acid and Ginger as a Potent Antioxidant

Vitamin C is one of the naturally occurring antioxidants capable of reducing or preventing skin photoaging. Achieving a stable formulation with the optimal dose of ascorbic acid to ensure a biologically significant antioxidant effect is a challenge when developing cosmetic formulations. The objective of this study was to develop a stable formula in a non-aqueous media with 15% pure vitamin C supplemented with ginger and to study its efficacy, skin tolerance, and cosmetic assessment in 33 women. Vitamin C stability over time was determined via a high-performance liquid chromatography (HPLC) technique versus an aqueous option. Reactive oxygen species (ROS) determination was quantified to provide antioxidant effect. A 56-day in vivo study was performed to evaluate skin luminosity and hyperpigmentation reduction. Skin acceptability was verified by a dermatologist. The HPLC studies demonstrated a high stability of the anhydrous formula compared to an aqueous option. The in vitro studies showed a reduction in ROS of 93% (p-value &lt; 0.0001). In vivo, luminosity increased by 17% (p-value &lt; 0.0001) and skin tone became 10% more uniform (p-value &lt; 0.007). Moreover, very good skin tolerance was determined as the dermatologist did not determine any clinical signs, and the subjects did not report any feelings of discomfort. We were able to develop an anhydrous formula of pure vitamin C that combines very good stability, consumer acceptance, and skin tolerance with a high level of efficacy.

Pharmacokinetics of isoniazid and rifapentine in young pediatric patients with latent tuberculosis infection

ObjectivesThis study investigated the steady-state pharmacokinetic profiles of 3-month weekly rifapentine plus isoniazid (3HP) in children with latent tuberculosisinfection (LTBI). We also assessed other factors, including tablet integrity, food, and pharmacogenetics. MethodsDuring the 3HP treatment, blood and urine samples were collected in week 4. Isoniazid and rifapentine levels were measured using a high-performance liquid chromatography technique. The genetic variation of arylamine N-acetyltransferase 2 (NAT2) and arylacetamide deacetylase (AADAC) were assessed by the MassARRAY®. Safety and clinical outcomes at week 48 were monitored. ResultsA total of 12 children with LTBI (age 3.8 [range 2.1-4.9 years old]) completed the treatment (isoniazid and rifapentine dose 25.0 [range 21.7-26.8] and 25.7 [range 20.7-32.1] mg/kg, respectively). No serious adverse events or active TB occurred. Tablet integrity was associated with decreased area under the concentration-time curve (91 vs 73 mg.h/l, P= 0.026) and increased apparent oral clearance of isoniazid (0.27 vs 0.32 l/h/kg, P= 0.019) and decreased rifapentine's renal clearance (CLR, 0.005 vs 0.003 l/h, P= 0.014). Food was associated with increased CLR of isoniazid (3.45 vs 8.95 l/h, P= 0.006) but not rifapentine. Variability in NAT2 and AADAC did not affect the pharmacokinetics of both drugs. ConclusionThere is high variability in the pharmacokinetic profiles of isoniazid and rifapentine in young children with LTBI. The variability was partly influenced by tablet integrity and food, but not pharmacogenetics. Further study in a larger cohort is warranted to display the relationship of these factors to treatment outcomes.

Antibacterial Activity and Components of the Methanol-Phase Extract from Rhizomes of Pharmacophagous Plant Alpinia officinarum Hance.

The rhizomes of Alpinia officinarum Hance (known as the smaller galangal) have been used as a traditional medicine for over 1000 years. Nevertheless, little research is available on the bacteriostatic activity of the herb rhizomes. In this study, we employed, for the first time, a chloroform and methanol extraction method to investigate the antibacterial activity and components of the rhizomes of A. officinarum Hance. The results showed that the growth of five species of pathogenic bacteria was significantly inhibited by the galangal methanol-phase extract (GMPE) (p < 0.05). The GMPE treatment changed the bacterial cell surface hydrophobicity, membrane fluidity and/or permeability. Comparative transcriptomic analyses revealed approximately eleven and ten significantly altered metabolic pathways in representative Gram-positive Staphylococcus aureus and Gram-negative Enterobacter sakazakii pathogens, respectively (p < 0.05), demonstrating different antibacterial action modes. The GMPE was separated further using a preparative high-performance liquid chromatography (Prep-HPLC) technique, and approximately 46 and 45 different compounds in two major component fractions (Fractions 1 and 4, respectively) were identified using ultra-HPLC combined with mass spectrometry (UHPLC-MS) techniques. o-Methoxy cinnamaldehyde (40.12%) and p-octopamine (62.64%) were the most abundant compounds in Fractions 1 and 4, respectively. The results of this study provide data for developing natural products from galangal rhizomes against common pathogenic bacteria.

Investigation the Correlation Between the Pathogenicity and Toxins Produced by Fusarium solani

In this study, 17 isolates of Fusarium solani has been isolated from the different geographic areas in Iraq, all isolates were identical to the species F. solani The results show a strong correlation between pathogenicity and toxins produced by F. solani. The results also showed that there were significant differences between these isolates. Isolate (S2Fs5) was superior in disease severity (98.50,96.10%) while isolate (K6Fs17.) were lower 90.20,88.10 as compared to control treatment 0%. High-performance liquid chromatography (HPLC) technique for F. solani culture filtrate showed that isolate (S2Fs5) higher production of toxin (2991.7, 3497.9) while isolate (K6FS17.) was lower production of toxin (317.5,211.3). All of the isolates produced different amount of T2 toxin and Deoxynivalenol, the content of the two toxins has been correlated with pathogenicity.

High Performance Liquid Chromatography: Recent Patents and Advancement

Background: Chromatography is defined as a set of techniques that are used for the separation of constituents in a mixture. Introduction: High-Pressure Liquid Chromatography or High-Performance Liquid Chromatography (HPLC) is known as a specialized technique in which columns as well as liquid chromatography are used in the separation, characterizationand investigation of the active moieties existing in the mixture. Objective: Current review focuses on the HPLC technique, including its principles, instrumentation, types, applications, advancements, and patents. Result: HPLC technique is important both for quantitative as well as qualitative analysis and is used for the evaluation of biological and pharmaceutical samples. It is the safest, most versatile, and fastest technique for chromatographic analysis in the field of quality control of drug components. In this review, the authors have also tried to summarize some of the advancements and recent patents in which the HPLC technique was used for the analysis. Conclusion: The article will help in understanding the role and importance of this analytical technique in the quality control of drugs and biologicals.

A Review on Analytical Method Development and Validation by High Performance Liquid Chromatography Technique

Chromatography, although primarily a separation technique, is mostly employed in chemical analysis in which High-performance liquid chromatography (HPLC) is an extremely versatile technique where analytes are separated by passage through a column packed with micro meter-sized particles. Now a day reversed-phase chromatography is the most commonly used separation technique in HPLC. The reasons for this include the simplicity, versatility, and scope of the reversed-phase method as it is able to handle compounds of a diverse polarity and molecular mass. Reversed phase chromatography has found both analytical and preparative applications in the area of biochemical separation and purification. Molecules that possess some degree of hydrophobic character, such as proteins, peptides and nucleic acids, can be separated by reversed phase chromatography with excellent recovery and resolution. This review covers the importance of RP-HPLC in analytical method development and their strategies along with brief knowledge of critical chromatographic parameters need to be optimized for an efficient method development

Determination of valsartan in its pharmaceutical preparations using high-performance liquid chromatography technique

High-performance liquid chromatography technique was used to estimate valsartan in its pure form and in some pharmaceutical preparations. It was found through the results obtained that the proposed method is sensitive and accurate, and the estimation was done in a short time. In this work, a No L1, 150mm separation column was used to separate the components as they were type The UV spectrometer was used as a wavelength of 239 nm, the column temperature was 40°C, and the mobile phase consisted of 50:50 (acetonitrile: methanol: drops of triethylamine, pH = 2.4) at a flow rate of 1 ml/min. Through what was reached, we find that the method is sensitive, accurate and compatible, as the percentage recovery reached (100.63-103.35), and the linearity was (40-120 mcg/ml) for valsartan. The method was applied to a number of pharmaceutical preparations available in the local market, and the method was successful in the quantitative estimation of valsartan.

Use of high-performance liquid chromatography technique for the simultaneous determination of amlodipine and valsartan

This technique was used in the estimation of amlodipine and valsartan simultaneously. The method was sensitive and accurate, and the estimation was done in a short time. In this work, a separation column of type L1, 150 mm was used to separate and sold. The group also found the ultraviolet spectrophotometer that used a wavelength of 239 nm, the temperature of the column was 40 degrees Celsius, and the mobile phase, consisting of 50:50 (acetonitrile: methanol: drops of triethylamine, pH = 2.4) was long. Through what was reached, we find that the method is sensitive, accurate and medium in size (101.43-99.13) and (100.17-99.53), and linear punches (6-14 mcg/ml) and (48-112 mcg/ml) for the two drugs, respectively. The method was applied to a number of pharmaceutical preparations available in the local market, successful in the real-time quantification of amlodipine and valsartan, respectively.

Development and Validation of Analytical RP HPLC Method for Benidipine Hydrochloride

The aim of the present study is development and validation of analytical reverse phase (RP) high performance liquid chromatography (HPLC) from Benidipine Hydrochloride. HPLC method is used for quantification, identification and purification of particular analyte or compound. The developed HPLC chromatographic technique was validated based on international conference on harmonization (ICH) Q2 guidelines for accuracy, precision, linearity, range and robustness. High performance liquid chromatography technique is rapid and precise technique. Advance for the validated of Benidipine Hydrochloride in pure and tablet dosage form. Diethyl ether was extracted from plasma in the presence of 5M NaOH (Sodium Hydroxide) Diethyl ether is used as internal standard like Benidipine and Benidipine d5. Benidipine is dihydropyridine calcium channel blocker, which is used in the treatment of hypertension and angina pectoris and is used to examine the efficacy and safety of therapy with Benidipine in elderly hypertensive patients.

Measuring the Systemic Inflammatory Response to On- and Off-Pump Coronary Artery Bypass Graft (CABG) Surgeries Using the Tryptophan/Kynurenine Pathway

Background: Cardiac surgeries induce many inflammatory responses with remarkable clinical implications. Tryptophan (Trp) is a precursor for serotonin, melatonin and kynurenine (Kyn). Plasma kynurenic acid (Kyna) and Kyn concentrations are thought to be related to the severity of inflammation. Plasma Trp/Kyn ratio is used to measure inflammatory cytokine activity. Methods: We performed the current longitudinal study in a tertiary care center and included 62 patients divided into two groups; group A (on-pump CABG patients) and group B (off-pump CABG patients). Plasma Trp and Kyn were measured using the high-performance liquid chromatography (HPLC) technique. Serum interlukin-6 (IL-6) and white blood cells (WBCs) were measured using ELISA and routine blood count, respectively. Results: The present study revealed that the intraoperative levels of plasma Kyn, IL-6 and WBCs were significantly increased while the plasma Trp/Kyn ratio was significantly decreased in both the groups; however, the changes were more significant in the on-pump CABG group. Moreover, the levels in both the groups returned to preoperative levels 72 h postoperative. Our study has shown that WBCs is positively correlated with IL-6, but has negative correlation with Trp/Kyn ratio. Conclusions: Kyn and Trp/Kyn ratio might be utilized as markers of the severity of inflammation in major surgery. In addition, off-pump CABG might be more preferable than on-pump CABG regarding stress and release of inflammatory markers.

Korean clinical practice guidelines for the diagnosis of hereditary hemolytic anemia.

Although the prevalence of hereditary hemolytic anemia (HHA) is relatively low in Korea, it has been gradually increasing in recent decades due to increment in the proportions of hemoglobinopathies from immigrants of South East Asia, raising awareness of the disease among clinicians, and advances in diagnostic technology. As such, the red blood cell (RBC) Disorder Working Party (WP), previously called HHA WP, of the Korean Society of Hematology (KSH) developed the Korean Standard Operating Procedures (SOPs) for the diagnosis of HHA in 2007. These SOPs have been continuously revised and updated following advances in diagnostic technology [e.g., flow cytometric osmotic fragility test (FOFT) and eosin-5-maleimide (EMA) binding test], current methods for membrane protein or enzyme analysis [e.g., liquid chromatography-tandem mass spectrometry (LC-MS/MS), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), high-performance liquid chromatography (HPLC)], and molecular genetic tests using next-generation sequencing (NGS). However, the diagnosis and treatment of HHA remain challenging as they require considerable experience and understanding of the disease. Therefore, in this new Korean Clinical Practice Guidelines for the Diagnosis of HHA, on behalf of the RBC Disorder WP of KSH, updated guidelines to approach patients suspected of HHA are summarized. NGS is proposed to perform prior to membrane protein or enzyme analysis by LC-MS/MS, UPLC-MS/MS or HPLC techniques due to the availability of gene testing in more laboratories in Korea. We hope that this guideline will be helpful for clinicians in making diagnostic decisions for patients with HHA in Korea.

Production of Prosaikogenin F, Prosaikogenin G, Saikogenin F and Saikogenin G by the Recombinant Enzymatic Hydrolysis of Saikosaponin and their Anti-Cancer Effect.

The saponins of Bupleurum falcatum L., saikosaponins, are the major components responsible for its pharmacological and biological activities. However, the anti-cancer effects of prosaikogenin and saikogenin, which are glycoside hydrolyzed saikosaponins, are still unknown due to its rarity in plants. In this study, we applied two recombinant glycoside hydrolases that exhibit glycoside cleavage activity with saikosaponins. The two enzymes, BglPm and BglLk, were cloned from Paenibacillus mucilaginosus and Lactobacillus koreensis, and exhibited good activity between 30–37 °C and pH 6.5–7.0. Saikosaponin A and D were purified and obtained from the crude B. falcatum L. extract using preparative high performance liquid chromatography technique. Saikosaponin A and D were converted into saikogenin F via prosaikogenin F, and saikogenin G via prosaikogenin G using enzyme transformation with high β-glycosidase activity. The two saikogenin and two prosaikogenin compounds were purified using a silica column to obtain 78.1, 62.4, 8.3, and 7.5 mg of prosaikogenin F, prosaikogenin G, saikogenin F, and saikogenin G, respectively, each with 98% purity. The anti-cancer effect of the six highly purified saikosaponins was investigated in the human colon cancer cell line HCT 116. The results suggested that saikosaponins and prosaikogenins markedly inhibit the growth of the cancer cell line. Thus, this enzymatic technology could significantly improve the production of saponin metabolites of B. falcatum L.

Gall Nuts Cynips quercusfolii (Linnaeus) and Andricus infectorius (Hartig) as Tannin Raw Materials

The study included gall nuts caused by Cynips quercusfolii collected from Quercus robur and Andricus infectorius, harvested from Quercus infectoria. The evaluation of the percentage content of tannins expressed as a pyrogallol was performed using the analytical methodology recommended by the current “European Pharmacopoeia 10”. In order to compare the chemical composition of the tested samples, chromatographic profiles and ion mass distribution spectra were made using high-performance liquid chromatography techniques coupled with two types of detection: spectrophotometry and mass spectrometry. The average content of tannins expressed as pyrogallol for C. quercusfolii ranged from 13.36% to 14.74%, while for A. infectorius it was from 34.77% to 39.95%. The comparison of both the mass spectra and the chromatographic profiles shows a high similarity of all samples of C. quercusfolii and large differences in the chemical composition compared to A. infectorius. The tested C. quercusfolii should be considered a much poorer tannin source than A. infectorius. It cannot be simultaneously treated as its substitute raw material due to the lower tannins percentage content and different chemical composition. However, compared to other Central European herbal raw materials, C. quercusfolii gall nuts are an alternative tannin source with potential use in herbal medicine and the pharmaceutical industry.

A Sensitive Spectrofluorimetric Method for Curcumin Analysis

Curcumin (CUR), a natural polyphenolic compound extracted from the rhizomes of Curcuma longa, is used as a pharmaceutical agent, spice in food, and as a dye. Currently, CUR is being investigated for cancer treatment in Phase-II clinical trials. CUR also possesses excellent activities like anti-inflammatory, anti-microbial, and anti-oxidant, therefore quality control is crucial. The present research work was to develop a new, simple, validated and time-saving rapid 96-well plate spectrofluorimetric method for the determination of CUR. The developed method was compared with routinely used high performance liquid chromatography (HPLC) technique. The developed method were found to be linear in the concentration range of 15 to 3900 ng/mL with R2 ≥ 0.9983 for spectrofluorimetric and 50-7500 ng/mL with R2 ≥ 0.9999 for HPLC method. Accuracy, intraday and interday precision was adequate, with RSD lower than the suggested limits. The limits for the detection and the quantification of CUR were 7 and 15 ng/mL for spectrofluorimetric, and 25 and 50 ng/mL for HPLC respectively. The Bland-Altman analysis demonstrated the similarities between the two methods. The 96-well plate method was successfully applied to determine CUR in solid lipid nanoparticles (SLNs) and chitosan nanoparticles (Chi-NPs). The developed spectrofluorimetric method can hence serve as a possible replacement for the HPLC method for the quantification of CUR in healthcare and food products.

Comparative qualitative and quantitative assessment of selected brands of Co-trimoxazole tablets and suspensions procured from Lagos State, Nigeria using first derivative UV spectroscopy and HPLC

Background: The use of High-Performance Liquid Chromatography (HPLC) to evaluate the quality of co-trimoxazole formulations is relatively expensive, and First Derivative UV Spectroscopic (FDS) technique may be alternatively used. There are no studies evaluating the quality of locally manufactured sulphamethoxazole/trimethoprim (SMX/TMP) dosage forms procured from Lagos, Nigeria. The aim of the work was to evaluate the quality attributes of selected co-trimoxazole tablets and suspensions marketed in Lagos, Nigeria, and determine their API content using first derivative UV spectroscopy and HPLC. Methods: Ten samples each of Co-trimoxazole tablets and suspensions were procured and their quality attributes were evaluated using standard pharmacopeial methods while their drug content was quantified using FDS and HPLC methods. Results: 70 % of the tested tablet as well as all the suspension samples exhibited satisfactory quality attributes. In terms of the drug content assay, 60 % of the studied tablets and 40 % of the suspension brands were USP-compliant. The SMX and TMP content of the suspension formulations using FDS technique was generally less than that obtained using the HPLC method, suggesting that the active ingredients might have degraded. Conversely, there was good correlation in the drug content of the tablets using FDS and HPLC. Conclusion: First Derivative UV spectroscopy may be suitable to quantify the drug content of co-trimoxazole tablets while HPLC technique may be preferable for co-trimoxazole suspensions. Furthermore, stringent regulatory monitoring of the marketed co-trimoxazole products is required to assure the quality, safety and efficacy of marketed co-trimoxazole products, thereby safeguarding public health.

Morphological and Molecular Characterization of Some Alternaria Species Isolated from Tomato Fruits Concerning Mycotoxin Production and Polyketide Synthase Genes.

Tomatoes (Lycopersicon esculentum) are one of the main crops grown in Egypt. The fungal black spot illness of fruits is usually associated with the secretion of mycotoxin by Alternaria toxigenic species. Twenty Alternaria isolates were isolated from infected tomatoes fruits by baiting technique, morphologically identified to species level, and confirmed using Internal Transcribed Spacer (ITS) gene sequencing. ITS gene sequencing of fragments obtained 547, 547, 542, 554, and 547 bp for A. alternata, A. brassicicola, A. citri, A. radicina, and A. tenuissima, respectively. Alternaria species were investigated for mycotoxin production using the high-performance liquid chromatography (HPLC) technique. The data from the HPLC analysis showed that the mycotoxins were determined in four out of five Alternaria species, with the incidence ranging from 0.89–9.85 µg/mL of fungal extract at different retention times. Alternaria alternata was the most active species and produced three types of toxins. Polyketide synthase genes (pksH and pksJ) which are involved in the Alternaria toxin’s biosynthesis were also amplified from the DNA of Alternaria species.

Development and validation of stability-indicating RP-HPLC method for the estimation of azoxystrobin in its formulations

The current work established and validated a simple, selective, precise, and accurate HighPerformance Liquid Chromatographic technique (HPLC) for the analysis of Azoxystrobin in its formulations. The mobile phase is made up of a combination of mobile phases comprising Acetonitrile and water in proportion, 80:20 (v/v). At a run duration of 15 minutes, this was found to yield a sharp peak of Azoxystrobin. Azoxystrobin was analysed using HPLC at a wavelength of 255 nm at a flow rate of 1.0 mL/min. The calibration curve's linear regression analysis results revealed a satisfactory linear connection with a regression coefficient of 0.999 in the concentration range of 50% to 150 %. The linear regression equation was y = 2025x +123.2.The proposed approach was used to analyse Azoxystrobin with a high degree of precision and accuracy.The method was validated for precision, accuracy, specificity, ruggedness and robustness. This method is useful for the quantification of Azoxystrobin because of its precision, accuracy, short retention duration, sensitivity, and mobile phase composition.

Isolation and molecular characterization of nutritionally potent Arthrospira maxima from Indian paddy field

Arthrospira maxima (A. maxima) is a cyanobacterium renowned for its usage as food and feed supplements as a potential source of functional ingredients for developing nutraceutical foods of natural origin. The Indian paddy fields, which can naturally produce eco-friendly cyanobacterial mats soon after receiving monsoon rain, were studied for isolation and molecular confirmation of Arthrospira and exploration of its nutritional profile. First, the collected cyanobacterial mat was processed for isolation of pure culture. After that, axenic culture was maintained in Zarrouk's medium at 27 ± 2 °C under the photosynthetically active radiation (PAR) of 75 μmol photon m−2 s−1 with a 16/8 h of light/dark period in laboratory condition. Morphologically proven pure culture of A. maxima was subsequently analyzed for its genomic stability using Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) and confirmed by targeting 16S ribosomal RNA partial gene sequencing.Further confirmed and pure A. maxima extract was processed to find out its nutraceutical values (amino acids, fatty acid, vitamin and chlorophyll content) by using High-performance liquid chromatography (HPLC) and Gas chromatograph (GC) technique. Results showed that identified strain was confirmed as A. maxima, and its partial gene sequences had been submitted to NCBI to get a unique accession number (GenBank: KM407008.1.). The study explores the new strain of A. maxima as an habitant from the Indian paddy field; it may be considered a potential candidate for industrial biomass production at a large scale.

Thiamine Level in Out-of-hospital Cardiac Arrest Patients

BACKGROUND: Thiamine deficiency is more common in critically ill patients. Administration of thiamine in cardiac arrest mice has improved survival and neurological outcomes. Evidence for thiamine deficiency in cardiac arrest humans is insufficient to support routine use of thiamine in cardiac arrest patients. AIM: This study aimed to determine thiamine blood levels in cardiac arrest patients to understand whether the presence of thiamine deficiency is common in cardiac arrest patients. METHODS: A prospective descriptive study from April 2017 to March 2018, on 24 adult out-of-hospital cardiac arrest patients. We used the high-performance liquid chromatography technique to determine whole blood thiamine pyrophosphate levels in cardiac arrest patients who arrived at the emergency department within 1 h of the onset of a cardiac arrest. RESULTS: The mean thiamine pyrophosphate level within 1 h of the onset of a cardiac arrest was 170.9 ± 56.7 nmol/L. Only one participant had thiamine deficiency according to the cut-off level for thiamine pyrophosphate in whole blood of &lt;70 nmol/L. Fourteen patients had spontaneous return of circulation. Thiamine pyrophosphate levels were not different between the two groups of patients who had and did not have the return of spontaneous circulation at the emergency department. CONCLUSION: Little evidence was found to support thiamine deficiency as a feature among our cardiac arrest patients. A study with a larger population is required for more meaningful statistical analysis. As there is no consensus on cut-off level for thiamine deficiency diagnosis, the level of thiamine pyrophosphate in specific populations should be evaluated to establish reference values.