[Establishment of collection methods and laboratory testing methods for active pharmaceutical ingredient in the workplace air].

Objective: To establish collection methods and laboratory testing methods for qualitative and quantitative analysis of 9 typical active pharmaceutical ingredient in the workplace air. Methods: In December 2021, a mixed solution of nine analytes was prepared and then dispersed in aerosol state to simulate sampling. Glass fiber filter membrane was selected as air collector and collected active pharmaceutical ingredient in the air at a rate of 2.0 L/min for 15 minutes. Then, the obtained filter membrane samples were eluted with 25%ACN/75%MeOH. Finally, the eluent was qualitatively and quantitatively analyzed with liquid chromatography-triple quadrupole mass spectrometer. Results: This method could effectively collect active pharmaceutical ingredient in the air, with an average sampling efficiency of more than 98.5%. The linear correlation coefficient r was greater than 0.9990. The lower limit of quantification for each analyte ranged from 0.6~500.0 ng/ml, and the average recovery rate ranged from 97.6%~102.5%. Conclusion: This method could simultaneously collect 9 active pharmaceutical ingredient in the workplace air, and could provide accurate qualitative and quantitative analysis in subsequent laboratory tests.

Development of an eco-friendly fluorescent probe for mefenamic acid sensing in pharmaceuticals and biofluids.

Mefenamic acid, renowned for its analgesic properties, stands as a reliable choice for alleviating mild to moderate pain. However, its versatility extends beyond pain relief, with ongoing research unveiling its promising therapeutic potential across diverse domains. A straightforward, environmentally friendly, and sensitive spectrofluorometric technique has been developed for the precise quantification of the analgesic medication, mefenamic acid. This method relies on the immediate reduction of fluorescence emitted by a probe upon interaction with varying concentrations of the drug. The fluorescent probe utilized, N-phenyl-1-naphthylamine (NPNA), was synthesized in a single step, and the fluorescence intensities were measured at 480 nm using synchronous fluorescence spectroscopy with a wavelength difference of 200 nm. Temperature variations and lifetime studies indicated that the quenching process was static. The calibration curve exhibited linearity within the concentration range of 0.50-9.00 µg/mL, with a detection limit of 60.00 ng/mL. Various experimental parameters affecting the quenching process were meticulously examined and optimized. The proposed technique was successfully applied to determine mefenamic acid in pharmaceutical formulations, plasma, and urine, yielding excellent recoveries ranging from 98% to 100.5%. The greenness of the developed method was evaluated using three metrics: the Analytical Eco-scale, AGREE, and the Green Analytical Procedure Index.

Occurrence and risks of pharmaceuticals, personal care products, and endocrine-disrupting compounds in Chinese surface waters.

The continuous and rapid increase of chemical pollution in surface waters has become a pressing and widely recognized global concern. As emerging contaminants (ECs) in surface waters, pharmaceutical and personal care products (PPCPs), and endocrine-disrupting compounds (EDCs) have attracted considerable attention due to their wide occurrence and potential threat to human health. Therefore, a comprehensive understanding of the occurrence and risks of ECs in Chinese surface waters is urgently required. This study summarizes and assesses the environmental occurrence concentrations and ecological risks of 42 pharmaceuticals, 15 personal care products (PCPs), and 20 EDCs frequently detected in Chinese surface waters. The ECs were primarily detected in China's densely populated and highly industrialized regions. Most detected PPCPs and EDCs had concentrations between ng/L to µg/L, whereas norfloxacin, caffeine, and erythromycin had relatively high contamination levels, even exceeding 2000 ng/L. Risk evaluation based on the risk quotient method revealed that 34 PPCPs and EDCs in Chinese surface waters did not pose a significant risk, whereas 4-nonylphenol, 4-tert-octylphenol, 17α-ethinyl estradiol, 17ß-estradiol, and triclocarban did. This review provides a comprehensive summary of the occurrence and associated hazards of typical PPCPs and EDCs in Chinese surface waters over the past decade, and will aid in the regulation and control of these ECs in Chinese surface waters.

Occurrence of pharmaceuticals in Latin America: case study on hazard assessment and prioritization in Costa Rica.

Pharmaceuticals are considered as contaminants of emerging concern, and their occurrence in diverse environmental matrices has been described during the last 25 years. Nonetheless, pharmaceutical occurrence has not been evenly described worldwide, and reports from some geographical areas such as most parts of Latin America are scarce. This work aims to address the situation of water pollution due to pharmaceuticals in Latin America by means of two main goals: i. First, reviewing the monitoring studies performed in Latin America on this topic (period 2009-2024), which were conducted in Brazil, Mexico, Colombia, Ecuador, Peru and Argentina, to highlight the most frequently detected compounds from each therapeutic group in the region. ii. Second, analyzing the case of Costa Rica through the hazard assessment and prioritization of pharmaceuticals based on the monitoring performed in this country (years 2011; 2018-2019). The monitoring in Costa Rica comprised a total of 163 sampling points: wastewater treatment plants (WWTPs) (14 urban WWTPs plus two landfill WWTPs; total samples n = 44 influents and n = 34 effluents), nine hospital effluents (n = 32), wastewater from livestock farms (six swine farms and seven dairy farms; n = 23 influents and n = 37 effluents), 64 continental surface water sampling points (n = 137), and 61 coastal seawater sampling points (n = 61). Risk assessment of detected concentrations by the hazard quotient (HQ) approach (period 2018-2019) revealed a total of 25 medium or high-hazard compounds (out of 37 detected compounds). The prioritization approach (which included the Frequency of Appearance (FoA), the Frequency of PNEC exceedance (FoE), and the Extent of predicted no-effect concentration (PNEC) exceedance (EoE)), showed a critical list of nine pharmaceuticals: caffeine, diphenhydramine, acetaminophen, lovastatin, gemfibrozil, ciprofloxacin, ibuprofen, doxycycline and norfloxacin. These compounds should be taken into account as a first concern during the implementation of environmental policies related to pharmaceutical products in the region.

A Novel Apparatus for the Fully Automated Extraction and Online Liquid Chromatographic Analysis of Solid Environmental Samples: Application to the Pressurized Hot Water Extraction of Pharmaceuticals in Soil.

A new self-assembled apparatus for the extraction of solid samples was designed and implemented to perform a recirculated pressurized hot water extraction (R-PHWE) directly coupled to liquid chromatography-tandem mass spectrometry. To investigate the potential of this new extraction apparatus, 34 target pharmaceutical compounds were analyzed in loam, silt-loam, and silty-clay-loam soils. The target analytes were characterized by heterogeneous physicochemical properties (e.g., -1.60 ≤ log D ≤ 5.91 at pH = 7.2, i.e., at the mean pH values of the three soils). Design of experiments (DoE) was used to identify the best extraction conditions for the target analytes by studying temperature, pressure, and number of extraction cycles. The results of DoE optimization pointed out the significant influence of the number of cycles on recovery. The application of DoE set point to the three reference soils provided recoveries ≥60% for 21-25 out the 34 target analytes, depending on soil. Good recovery precision (<25%) and moderate suppressive matrix effect (≤40%) were found for most target analytes, regardless of the soil considered. The optimized R-PHWE procedure evidenced statistically higher recoveries for 16 out of 34 target analytes when compared to conventional off-line dynamic PHWE.

In Silico Assessment of Biomolecule Reactivity with Leachables.

Leachables in pharmaceutical products may react with biomolecule active pharmaceutical ingredients (APIs), for example, monoclonal antibodies (mAb), peptides, and ribonucleic acids (RNA), potentially compromising product safety and efficacy or impacting quality attributes. This investigation explored a series of in silico models to screen extractables and leachables to assess their possible reactivity with biomolecules. These in silico models were applied to collections of known leachables to identify functional and structural chemical classes likely to be flagged by these in silico approaches. Flagged leachable functional classes included antimicrobials, colorants, and film-forming agents, whereas specific chemical classes included epoxides, acrylates, and quinones. In addition, a dataset of 22 leachables with experimental data indicating their interaction with insulin glargine was used to evaluate whether one or more in silico methods are fit-for-purpose as a preliminary screen for assessing this biomolecule reactivity. Analysis of the data showed that the sensitivity of an in silico screen using multiple methodologies was 80%-90% and the specificity was 58%-92%. A workflow supporting the use of in silico methods in this field is proposed based on both the results from this assessment and best practices in the field of computational modeling and quality risk management.

Clarifications on the Intended Use of USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests.

In the execution of its legislated responsibilities, the United States Food and Drug Administration commonly refers to standard test methods detailed in the United States Pharmacopeia (USP). Microbiological test methods (contained in general chapters) are listed in chapters <51> to <80> with details regarded as enforceable where referenced as a test method. USP <61> "Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests" is a globally harmonized chapter that has been successfully employed for the enumeration of microorganisms recoverable from nonsterile finished drug products. The content of USP <61> is not always scientifically principled nor emphatically understood by all pharmaceutical microbiologists. Consequently, misunderstanding and misapplication of USP <61> may result in analyses and assessments of microbiological quality that are flawed or erroneous. In this article, clarification is provided to assist the pharmaceutical microbiologist in the appropriate and intended use of USP <61>, including provision of details not always commonly known or understood.

Rapid Separation and Detection of Drugs in Complex Biological Matrix Using TD-CDI Mass Spectrometer.

The drug detection technology plays a pivotal role in the domains of pharmaceutical regulation and law enforcement. In this study, we introduce a method that combines thermal desorption corona discharge ionization (TD-CDI) with mass spectrometry for efficient drug detection. The TD-CDI module, characterized by its compact and simple design, enables the separation of analytes within seconds and real-time presentation of one or two analyte peaks on the mass spectrum most of the time, which reduces matrix interference and improves detection performance. Through experimental investigation, we studied the characteristics of TD-CDI for analyte separation and detection, even with the same mass number, and optimized the TD-CDI approach. TD-CDI-MS was employed for the rapid detection of drugs in various traditional medicine, food products, and human samples. Additionally, by utilizing TD-CDI for segmented hair direct analysis, it becomes possible to trace the drug usage cycle of individuals. This underscores the feasibility of the proposed analytical method within the realm of drug detection.

Microchip isotachophoresis for green and sustainable pharmaceutical quality control: Method validation and application to complex pharmaceutical formulations.

Universal microchip isotachophoresis (µITP) methods were developed for the determination of cationic and anionic macrocomponents (active pharmaceutical ingredients and counterions) in cardiovascular drugs marketed in salt form, amlodipine besylate and perindopril erbumine. The developed methods are characterized by low reagent and sample consumption, waste production and energy consumption, require only minimal sample preparation and provide fast analysis. The greenness of the proposed methods was assessed using AGREE. An internal standard addition was used to improve the quantitative parameters of µITP. The proposed methods were validated according to the ICH guideline. Linearity, precision, accuracy and specificity were evaluated for each of the studied analytes and all set validation criteria were met. Good linearity was observed in the presence of matrix and in the absence of matrix, with a correlation coefficient of at least 0.9993. The developed methods allowed precise and accurate determination of the studied analytes, the RSD of the quantitative and qualitative parameters were less than 1.5% and the recoveries ranged from 98 to 102%. The developed µITP methods were successfully applied to the determination of cationic and anionic macrocomponents in six commercially available pharmaceutical formulations.

Large-Scale Screening of Pharmaceutical Compounds to Explore the Application Space of On-Tissue MALDI and MALDI-2 Mass Spectrometry.

The successful application of matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in pharmaceutical research is strongly dependent on the detection of the drug of interest at physiologically relevant concentrations. Here we explored how insufficient sensitivity due to low ionization efficiency and/or the interaction of the drug molecule with the local biochemical environment of the tissue can be mitigated for many compound classes using the recently introduced MALDI-MSI coupled with laser-induced postionization, known as MALDI-2-MSI. Leveraging a MALDI-MSI screen of about 1,200 medicines/drug-like compounds from a broad range of medicinal application areas, we demonstrate a significant improvement in drug detection and the degree of sensitivity uplift by using MALDI-2 versus traditional MALDI. Our evaluation was made under simulated imaging conditions using liver homogenate sections as substrate, onto which the compounds were spotted to mimic biological conditions to the first order. To enable an evaluable detection by both MALDI and MALDI-2 for the majority of employed compounds, we spotted 1 µL of a 10 mM solution using a spotting robot and performed our experiments with a Bruker timsTOF fleX MALDI-2 instrument in both positive and negative ion modes. Specifically, we demonstrate using a large cohort of drug-like compounds that ∼60% of the tested compounds showed a more than 10-fold increase in signal intensity and ∼16% showed a more than 100-fold increase upon use of MALDI-2 postionization. Such increases in sensitivity could help advance pharmaceutical MALDI-MSI applications toward the single-cell level.

Quantitative 31P NMR Spectroscopy Platform Method for the Assay of Oligonucleotides as Pure Drug Substances and in Drug Product Formulations Using the Internal Standard Method.

One of the most widely used techniques for the quantification of small interfering ribonucleic acid (siRNA) is the ultraviolet (UV) spectroscopy method. However, due to uncertainties in the extinction coefficient affecting the accuracy of the method and a sample preparation including several dilution steps, the purpose of this study was to explore the possibility of determining the content of siRNA by a platform method using quantitative 31P nuclear magnetic resonance (31P-qNMR) and the internal standard method. In this paper, acquisition time, selection of a suitable internal certified reference material, signal selection used for quantification, relaxation delay, and precision are discussed. In addition, the robustness of the method and the ability to apply this platform method to both drug substance (DS) and drug product samples is also discussed. Quantifications of siRNA determined by the 31P-qNMR platform method were on average 98.5%w/w when adjusting for the sodium and water contents. The data confirmed the applicability of 31P-qNMR in siRNA content determinations. The quantifications were compared to quantifications determined by the traditional UV spectroscopy method by F- and t-tests. The statistical tests showed that the platform 31P-qNMR method provided more accurate results (mass balance close to 100% w/w) compared to the traditional UV spectroscopy method when analyzing DS samples.

Assessing the pharmaceutical residues as hotspots of the main rivers of Catalonia, Spain.

The global increase in pharmaceutical consumption, driven by factors such as aging populations and chronic diseases, has raised concerns regarding the environmental impact of pharmaceutical contaminants. Europe, and more specifically Catalonia (Spain), exhibits high pharmaceutical consumption rates, potentially exacerbating environmental contamination. Pharmaceuticals enter rivers through various pathways, persisting after wastewater treatment plants and posing risks to aquatic organisms and human health. Llobregat and Besòs Rivers in Catalonia, crucial water sources, demonstrate detectable pharmaceutical levels, necessitating comprehensive analysis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) proves effective in detecting pharmaceutical residues, facilitating their risk assessment. This paper reviews the occurrence, fate, and risks associated with 78 pharmaceuticals and metabolite in Llobregat and Besòs Rivers, using LC-MS/MS for analysis. Understanding pharmaceutical impacts on Catalonian River ecosystems is essential for developing mitigation strategies.

[Research Progress on Pollution Characteristics, Degradation, and Transformation of Typical PPCPs in the Process of Wastewater Treatment].

Pharmaceuticals and personal care products (PPCPs) have received extensive attention as a new type of pollutant inin the 21st century, and the ecological and health risks caused by PPCPs have gradually been recognized by government regulatory agencies. Daily use of PPCPs has led to their frequent detection and high concentrations in the influent, effluent, and sludge of wastewater treatment plants, but traditional wastewater treatment processes can't remove them effectively. Most research about enhancing the removal of PPCPs through microbial degradation, photodegradation, and ozonation is still in the laboratory research stage, and the removal effects are not satisfactory when applied to actual sewage treatment. Therefore, the effective removal of PPCPs from domestic wastewater is a critical technical problem that urgently needs to be studied and solved in the coming years. At present, many scholars do not have a comprehensive understanding about the degradation and transformation behaviors of microbes, ultraviolet, and ozone for typical PPCPs in the wastewater treatment process, so it is necessary to conduct a systematic analysis and discussion. In this study, 16 typical PPCPs frequently detected in sewage treatment plants were selected as research objects through a literature review. The occurrence, removal characteristics, and sludge adsorption properties of typical PPCPs in wastewater treatment plants were analyzed and summarized. The degradation and transformation behavior of typical PPCPs under microbial, ultraviolet, and ozone treatments in the wastewater treatment process were also discussed. Finally, based on current research gaps, some research directions for the removal and transformation of PPCPs in wastewater were proposed:① investigation into the removal characteristics of PPCPs by actual biochemical treatment; ② study on the mechanism of microbial degradation and transformation of typical PPCPs during biochemical treatment; ③ study on the degradation and transformation mechanism of typical PPCPs by UV/ozone in an actual sewage system; and ④ research on the application technology of removing PPCPs from sewage via microbial degradation, photodegradation, ozone oxidation, etc. The relevant results of this study can provide a reference for the pollution control of typical PPCPs in the sewage treatment process.

For the occurrence of PPCPs from source to tap: A novel approach modified in terms of sample preservation and SPE cartridge to monitor PPCPs in our water supply.

BACKGROUND: The ongoing infusion of pharmaceutical and personal care products (PPCPs) into ecosystems sustains a perpetual life cycle and leads to multi-generational exposures. Limited understanding of their environmental impact and their intrinsic ability to induce physiological effect in humans, even at low doses, pose great risks to human health. Few scholarly works have conducted systematic research into the occurrence of PPCPs within potable water systems. Concurrently, the associated monitoring techniques have not been comprehensively examined with regards to the specific nature of drinking water, namely whether the significant presence of disinfectants may influence the detection of PPCPs. RESULTS: A modified approach in terms of detailed investigation of sample preservation and optimization of an in-lab fabricated solid phase extraction (SPE) cartridge filled with DVB-VP and PS-DVB sorbent was proposed. Favorable methodological parameters were achieved, with correlation coefficients spanning from 0.9866 to 0.9998. The LODs of the PPCPs fluctuated from 0.001 to 2 µg L-1, while the LOQs varied from 0.002 to 5 µg L-1. The analysis of spiked samples disclosed a methodological precision of 2.31-9.86 % and a recovery of 52.4-119 %. We utilized the established method for analyzing 14 water samples of three categories (source water, finished water and tap water) from five centralized water supply plants. A total of 24 categories encompassing 72 PPCPs were detected, with the concentrations of PPCPs manifested a marked decrease from source water to finished water and finally to tap water. SIGNIFICANCE: Our research meticulously examined the enhancement and purification effects of widely used commercial SPE cartridges and suggested the use of in-lab fabricated SPE cartridges packed with DVB-VP and PS-DVB adsorbents. We also conducted a systematic evaluation of the need to incorporate ascorbic acid and sodium thiosulfate as preservatives for PPCP measurement, in consideration of the unique characteristics of drinking water matrices, specifically, the significant concentration levels of disinfectants. Furthermore, the proposed method was effectively employed to study the presence of PPCPs in source water, finished water, and tap water collected from centralized water supply plants.

Innovative Solid-Phase Extraction Strategies for Improving the Advanced Chromatographic Determination of Drugs in Challenging Biological Samples.

In the past few decades, considerable scientific strides have been made in the subject of drug analysis in human biological samples. However, the risk caused by incorrect drug plasma levels in patients still remains an important concern. This review paper attempts to investigate the advances made over the last ten years in common sample preparation techniques (SPT) for biological samples based on solid sorbents, including solid-phase extraction (SPE) and solid-phase micro-extraction (SPME), and in particular in the field of molecularly imprinted polymers (MIPs), including non-stimuli-responsive and stimuli-responsive adsorbents. This class of materials is known as 'smart adsorbents', exhibiting tailored responses to various stimuli such as magnetic fields, pH, temperature, and light. Details are provided on how these advanced SPT are changing the landscape of modern drug analysis in their coupling with liquid chromatography-mass spectrometry (LC-MS) analytical techniques, a general term that includes high-performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UHPLC), as well as any variation of MS, such as tandem (MS/MS), multiple-stage (MSn), and high-resolution (HRMS) mass spectrometry. Some notes are also provided on coupling with less-performing techniques, such as high-performance liquid chromatography with ultraviolet (HPLC-UV) and diode array detection (HPLC-DAD) detection. Finally, we provide a general review of the difficulties and benefits of the proposed approaches and the future prospects of this research area.

A Novel Low-Frequency Electromagnetic Active Inertial Sensor for Drug Detection.

The purpose of this paper is to demonstrate a new discovery regarding the interaction between materials and very low radio frequencies. Specifically, we observed a feedback response on an inertia active sensor when specific frequencies (around 2-4 kHz) are used to irradiate targeted pharmaceutical samples like aspirin or paracetamol drugs. The characteristics of this phenomenon, such as excitation and relaxation time, the relation between deceleration and a material's quantity, and signal amplitude, are presented and analyzed. Although the underlying physics of this phenomenon is not yet known, we have shown that it has potential applications in remote identification of compounds, detection, and location sensing, as well as identifying substances that exist in plants without the need for any processing. This method is fast, accurate, low-cost, non-destructive, and non-invasive, making it a valuable area for further research that could yield spectacular results in the future.

NMR Longitudinal Rotating Frame Relaxation Time (T1ρ) with a Weak Spin Locking Field as an Approach to Characterize Solid-State Active Pharmaceutical Ingredients: Proof of Concept.

Nuclear magnetic resonance (NMR) longitudinal rotating frame relaxation time (T1ρ), rarely used in low-field NMR, can be more effective than conventional T1 and T2 relaxation times to differentiate polymorphic forms of solid pharmaceuticals. This could be attributed to T1ρ sensibility to structural and molecular dynamics that can be enhanced by changing the strength of the oscillating magnetic field (B1) of spinlock pulses. Here, we compared the capacity of T1, T2, and T1ρ to differentiate inactive (A) and active (C) crystalline forms of the World Health Organization essential drug Mebendazole. The results showed that T1 and T2 values of both forms were statistically identical at 0.47 T. Conversely, T1ρ of both forms measured with weak spinlock B1 fields, ranging from 0.08 to 0.80 mT were statistically different in the same spectrometer. The T1ρ also has the limit of detection to detect the presence of at least 10% of inactive A form in the active C form. Therefore, T1ρ, measured with weak spinlock B1 fields can be an effective, streamlined, and complementary approach for characterizing not only solid active pharmaceutical ingredients but other solid-state materials as well.

A sustainable sensitive spectrofluorimetric approach for the determination of the multipotent protein lactoferrin in different pharmaceuticals and infant milk formula: Compliance with greenness metrics.

The current study presents the first spectrofluorimetric approach for the estimation of lactoferrin, depending on the measurement of its native fluorescence at 337 nm after excitation at 230 nm, without the need for any hazardous chemicals or reagents. It was found that the fluorescence intensity versus concentration calibration plot was linear over the concentration range of 0.1-10.0 µg/mL with quantitation and detection limits of 0.082 and 0.027 µg/mL, respectively. The method was accordingly validated according to the ICH recommendations. The developed method was applied for the estimation of lactoferrin in different dosage forms, including capsules and sachets with high percent recoveries (97.84-102.53) and low %RSD values (<1.95). Lactoferrin is one of the key nutrients in milk powder and a significant nutritional fortifier. In order to assess the quality of milk powder, it is essential to rapidly and accurately quantify the lactoferrin content of the product. Therefore, the presented study was successfully applied for the selective estimation of lactoferrin in milk powder with acceptable percent recoveries (96.45-104.92) and %RSD values (≤3.607). Finally, the green profile of the method was estimated using two assessment tools: Green Analytical Procedure Index (GAPI) and Analytical GREEnness (AGREE), which demonstrated its excellent greenness.

Occurrence of pharmaceutically active compounds in groundwater and their effects to the human health.

Groundwater contamination by pharmaceutically active compounds (PhACs) has been considered a public health concern worldwide. Alongside the potential toxicological risk of these organic substances, many countries still rely on groundwater for drinking water supply. Thus, this study identified a priority list of seven licit PhACs, comprising acetaminophen (ACT), tramadol (TRA), carbamazepine (CBZ), erythromycin (ERY), sulfamethoxazole (SMX), metformin (MET), and oxazepam (OXZ). Consumption, concentration, and human toxicity in silico results were collected from open access databases. These three indicators were analyzed separately and grouped through a general risk index. The consumption index (data from the USA and Brazil) indicated that ACT, TRA, and MET are the most consumed. Monitoring samples from the USA and Europe (n = 816) indicated that OXZ and ERY stand out as the higher occurrence index considering both regions, but the ranking for each region showed considerable differences. When assessing toxicological risk, an index ≥ 0.5 was attributed to CBZ, MET, OXZ, SMX, and TRA. The general risk indicated the need to be attentive to MET, OXZ, and TRA as they presented ≥ 0.5 index values for at least two indicators.