INTRODUCTION:

First of all, the GAC (Green Analytical Chemistry) is an approach where the preserving the sustainability of environment is the major focus. By applying various metrics that are being in use from the starting of the concept of GAC. Different metrics of GAC are as follows:

1. National Environmental Methods Index (NEMI)

2. Analytical Eco-Scale

3. Multi-criteria Decision Analysis (MCDA)

4. Green Analytical Procedures Index (GAPI)

5. Red, Green, Blue (RGB) additive colour model

6. AGREE: Analytical Greenness metric tool

Overview of AGREE prep:

The AGREE prep open access software can be obtained from mostwiedzy.pl/AGREE prep, and the code is available at git.pg.edu.pl/p174235/agree prep. In case of difficulties with deploying the desktop version of the software, the user can access an online emulator by following the link provided at the project’s website (mostwiedzy.pl/agree prep), where one can also find a link to a short video with instructions on how to run the emulator.

AGREE prep's twelve distinct assessment processes yield scores between 0 and 1, where the extremes correspond to the worst and best achievement, respectively. Assessors have the option to modify the default weights assigned to each criterion in order to better suit their analytical objectives, as long as they provide justification for the change. Each criterion's score is weighted and added together to create the overall score, which also goes from 0 to 1, where 1 denotes ideal performance.

After the evaluation is finished, the AGREE prep software generates a round pictogram with a circle in the middle that displays the overall score and twelve trapezoid bars that correspond to the twelve criteria, each of which has a length equal to the assigned weight. The software asks for input data for each of the twelve assessment steps. Each element's colour changes upon review, making it simple to determine the procedure's strong and weak points as well as how each affects the overall result.

The assessment criteria are then explained in detail, with case studies used to illustrate the many facets of the greenness assessment and examples of their input values.

Importance in Analytical chemistry:

1. Standardized Evaluation of Sample Preparation Greenness: AGREE prep provides a quantitative, harmonized evaluation of the greenness of sample preparation methods, based on the 10 principles of Green Sample Preparation (GSP), enabling clear and objective comparisons.

2. Visual Representation for Quick Assessment: It offers a color-coded pictogram (circular diagram) for each method evaluated, allowing researchers to quickly identify strengths and weaknesses in their approach across all 10 GSP principles.

3. Supports Method Optimization: By pinpointing specific areas of improvement (e.g., high solvent use, poor energy efficiency), AGREE prep guides the optimization of analytical methods toward greener alternatives.

4. Enhances Sustainable Method Development: Integrating AGREE prep into method development ensures environmental sustainability is considered from the earliest stages, promoting eco-friendly decision-making in research and industry.

5. Facilitates Regulatory and Compliance Alignment: The software supports laboratories in aligning with green chemistry guidelines, such as those advocated by ISO standards and regulatory authorities emphasizing sustainability.

6. User-Friendly Interface for Broader Adoption: With a simple, user-friendly interface, AGREE prep makes green evaluation accessible to chemists without requiring extensive training in environmental metrics.

7. Customizable and Transparent Scoring: AGREE prep allows users to adjust weights and parameters, offering flexibility and transparency in how greenness scores are calculated based on method-specific context.

8. Educational Tool for Green Chemistry Awareness: The software serves as a didactic aid, helping students and researchers understand how each aspect of sample preparation contributes to environmental impact.

9. Supports Publication and Peer Review Standards: Including AGREE prep evaluations in analytical method publications enhances scientific transparency and reproducibility, and may meet editorial requirements for green assessment.

10. Promotes Greener Innovation in Analytical Chemistry: By integrating green metrics into the method development lifecycle, AGREE prep fosters innovation in designing cleaner, safer, and more sustainable analytical protocols.

AGREE: Analytical Greenness Calculator

Principle 1: Direct analytical techniques should be applied to avoid sample treatment ¹

Figure 1. Principle 1

1 Remote Sensing without Sample Damage:

· Remote sensing without sample damage implies the non-invasive and non-destructive measurement of a sample's characteristics without physically contacting, consuming, or altering the sample

Focus:

· Do not require solvents, reagents, or destructive extraction steps.

· Allow real-time analysis or in-line monitoring.

· Preserve the integrity of the pharmaceutical product (e.g., tablets, capsules, injections).

Example – NIR Spectroscopy for Tablet Uniformity:

· A pharmaceutical company uses NIR spectroscopy to check API (active pharmaceutical ingredient) content in tablets on a production line.

· Tablets move under an NIR probe—no grinding, solvent, or extraction needed.

· The spectrometer remotely senses chemical information by detecting how the sample absorbs NIR light.

· The sample remains untouched—no damage.

2 Remote Sensing with a Little Physical Damage

· The technique allows remote detection of analytes with limited, acceptable disturbance to the sample.

· The integrity of the majority of the sample is preserved.

Example: Solid-Phase Microextraction (SPME) in Residual Solvent Analysis

· In Analyzing residual solvents in tablets, traditional GC methods require solvent extraction—destructive and environmentally hazardous.

SPME Approach:

· A small SPME fiber is exposed to the headspace of a vial containing the tablet.

· Only a small volatile fraction of the sample is absorbed.

· The fiber is inserted into a GC-MS injector.

3 Non-Invasive Analysis:

Focus:

· Do not require any physical contact with the sample,

· Do not alter, damage, or consume the sample in any way,

· Allow direct, in situ measurement—often remotely

· The use of optical or sensor-based methods that can analyze the chemical or physical properties of a sample without disturbing it.

Example: NIR Spectroscopy for Tablet Testing Through Blister Packs

· A QC department wants to verify active pharmaceutical ingredient (API) content in tablets without opening the packaging.

· Use portable NIR spectrometers that shine light through the blister pack directly onto the tablet.

· Light is reflected back and analyzed for chemical composition.

· No need to remove tablets or use any solvent.

4 In-Field Sampling and Direct Analysis:

· Performing sample collection and analysis at or near the point of origin,

· Eliminating the need to transport the sample to a centralized lab,

· Avoiding elaborate preparation, such as filtration, dilution, or chemical treatment.

Example: Portable Raman Spectroscopy for On-Site Drug Authentication

· Field officers in rural regions or customs departments want to check authenticity of medicines on the spot (e.g., to detect falsified or substandard drugs).

In-Field Approach:

· A handheld Raman spectrometer is used to scan tablets or capsules.

· Spectral fingerprint is compared instantly to a built-in database.

· No need for sample prep, no chemicals, no lab required.

5 In-Field Sampling and On-Line Analysis:

· Collecting samples directly in the production or application environment, and sending them immediately and automatically into analytical instruments that are connected in real-time (on-line) to the production process.

· Avoid transporting samples to the lab or extensive manual preparation.

Example: On-line NIR Monitoring of Blend Uniformity in Tablet Manufacturing

· A company produces tablets using a continuous blending process for APIs and excipients.

On-Line Setup:

· An NIR sensor is mounted inside the blender.

· As powders mix, the NIR probe continuously reads chemical composition.

· If blend uniformity falls below set limits, the process is auto-adjusted or halted.

6 On-Line Analysis:

· Are directly integrated into the production or reaction process,

· Perform real-time or continuous monitoring,

· Often include automated sampling and analysis,

· Provide immediate feedback for process control.

Example: On-Line UV-Vis for Drug Dissolution Monitoring

· During drug manufacturing, dissolution of an active compound must be monitored (e.g., API solubility in a reaction vessel).

On-Line Setup:

· A flow-through UV-V is detector is installed in the reaction line.

· The solution is continuously passed through a transparent flow cell.

· UV absorbance is measured without collecting or handling samples.

7 At-line Analysis:

· Samples are taken near the production process (close to the production line or process area)

· The operator manually or semi-automatically prepares and analyzes the sample right away,

· The goal is rapid feedback with minimal delay but not fully real-time or automated like on-line analysis.

Example: At-Line NIR for Moisture Content in Granules;

· During granulation, controlling moisture content is crucial.

· Samples are grabbed from the granulator and taken to a nearby NIR station.

At-Line Setup:

· The sample is analyzed immediately with NIR spectroscopy.

· Results are obtained quickly (within minutes) to adjust drying parameters if needed.

8 Off-line analysis:

· Samples are collected and physically removed from the production or process environment,

· Taken to a separate laboratory or analytical facility for testing,

· Typically involves manual sample preparation (grinding, dilution, extraction, etc.),

· Analysis is not immediate; there can be delays due to transport, preparation, and lab scheduling,

· The process is separate from the production line and not automated.

Example: Off-Line HPLC Analysis for API Quantification:

· A batch of tablets is produced, and a sample is taken from the batch.

· The sample is transported to the QC lab.

· Sample tablets are crushed, dissolved in solvents, and filtered before injection into HPLC.

9 External Sample Pre- and Treatment and Batch Analysis (Reduced Number of Steps):

· External sample pre- and treatment means that sample preparation (like extraction, filtration, dilution, or cleanup) happens outside the main production or analytical system, typically in a separate lab or preparation area.

· Batch analysis means samples are prepared and analyzed in groups (batches), rather than continuously or one-by-one.

· The phrase "reduced number of steps" indicates a streamlined, simplified sample preparation process, minimizing time, reagents, and manual handling.

Example: Batch HPLC Analysis with Simplified Sample Prep for Tablet Assay:

· Tablets from a batch are sampled and sent to the QC lab.

· Instead of multiple complex extraction steps, samples are simply crushed and dissolved directly in a solvent.

· A batch of prepared samples is analyzed by HPLC sequentially.

· Fewer preparation steps reduce solvent use and lab time.

10 External Sample Pre- and Treatment and Batch Analysis (Large Number of Steps):

· A large number of steps means the sample preparation involves multiple, often complex and time-consuming procedures, such as extraction, purification, concentration, filtration, dilution, derivatization, etc.

Example: Multi-Step Extraction and HPLC Analysis for Trace Impurities:

· Detecting trace-level impurities or degradation products in a drug batch.

· Sample undergoes solvent extraction to isolate impurities,

· Followed by filtration, evaporation to concentrate,

· Derivatization to improve detection,

· Finally batch-analysed by HPLC or GC.

· The first one, which received the highest score, is in-line/in situ sample preparation, which takes into account sample preparation that is done immediately in the item under investigation without sample withdrawal.

· The second method is called "online/in situ sample preparation," which involves sampling and sample preparation in a side-line of the object being studied. Samples are continually taken from the object and then produced in situ using an automated system. Such online systems are typically not commercially available, and each application requires a unique interface². Note that online/in situ analysis should not be confused with laboratory-based online/at-line analysis, which consists of systems that combine sample preparation with the resulting analysis method³.

· The third method is on-site sample preparation, which entails gathering samples there and preparing them with the sample preparation device or devices that were transported to the sampling location. After that, the extracts are brought to the lab for additional examination.

Samples are taken from the object and brought to the lab for processing and analysis in the final ex situ sample preparation option. With the lowest score, this option represents the worst-case situation⁴.

Principle 2: Minimal sample size⁵and minimal number of samples are preferred⁶

Figure 2. Principle 2

Score = -0.142 × in (amount of sample in g or mL) + 0.65

· Every chemical that is harmful through any exposure pathway is listed here, and AGREE prep believes that any quantity greater than 10 millilitres or grams is unacceptable (score 0). In actuality, certain processes—like bringing the pH down to a certain level—do not specify how much of each reagent is utilised. In these situations, the type of reagent and sample size can be taken into account, and then personal laboratory expertise can be used to estimate these amounts.

· Water, inert gases, environmentally friendly substitutes for dangerous chemicals, and substances that are permitted for ingestion other than ethanol are all regarded as safe chemicals. A previous study examined a number of safe and natural reagent options, such as enzymes or unrefined plant extracts⁷.

· Furthermore, alcohols, esters, carboxylic acids, and terpenes were among the green and bio-based solvents that were recently compiled⁸.

· One of the most significant and plentiful renewable resources for a variety of sample preparation uses, such as paper strips and filter sheets, is cellulose. Cork, cotton, and other materials derived from living plants and animals that are powered by solar energy are further examples.

· Miniaturization and takes into account the sample size, which is stated in mass or volume units. Large samples may raise energy demands (during heating, cooling, and mineralizing), chemical consumption, and waste generation, in addition to lowering the potential for automation and mobility, even if sample size is typically not an issue from the perspective of abundance. Sample sizes more than 100 millilitres or grams are unacceptable for these reasons.

Principle 3: What is the positioning of the analytical device? (In-situ measurements):

Figure 3. Principle 3

Principle 6: Select derivatization agents (if used)

Figure 6. Principle 6

· "Select derivatization agents" refers to choosing a chemical reagent that can react with your target analyte to improve detectability, especially in techniques like GC, HPLC, or MS.

· Example: CAS No. 24277-44-9 corresponds to N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) – a widely used silylation agent.

· purpose: Converts polar functional groups (–OH, –COOH, –NH₂) into more volatile and thermally stable trimethylsilyl (TMS) derivatives

· Score of 1 is given when no derivatization is applied

Score = DA₁ × DA₂ ×... × DAn, -0.2

DA, is the score corresponding to the particular derivatization agent used

Principle 7: The Amount of Analytical Waste in g or mL⁹

Figure 7. Principle 7

Table 4: Amount of analytical waste

Score	Amount of waste (mL or g)
1	< 0.1
0.4	10
0.25	25
0	100

Score based on mass of waste is calculated as

Score = -0.134 × in (amount of waste in g or mL) +0.6946

(P) The number of consumables and single-use materials (such as filters, extraction thimbles, solid-phase extraction (SPE) cartridges, and micropipette tips) is measured in grams.

(Q) The ratio of the quantity of reusable materials (such solid-phase microextraction (SPME) fibres) to their maximum number of uses.

(R) Next, the quantity of chemicals needed to prepare the sample is taken into account. Since these factors are evaluated in later criteria, any reagents and solvents employed must be taken into account at this stage, regardless of any potential risks to the environment, human health, or safety.

(S) The amount of materials divided by the number of times they can be used must be subtracted in order to right amount (R).

· Depending on the sample preparation process, the sample itself (or a portion of it) may be deemed waste if the in line/in situ sample preparation methodology is not followed.

(T) When liquid samples are treated with reagents (such as acids, bases, or salts) or solvents, the entire amount of the sample is regarded as waste.

· A zero will be taken to waste from the sample is selected when

1. The sample is not treated with chemicals

2. The acceptor phase and the sample are not in touch (e.g., headspace methods) or

3. The sample is not contaminated by the acceptor phase (such as thin film microextraction (TFME), stir bar sorption extraction (SBSE), or direct-SPME).

· A 0% contribution to waste is regarded when solid samples are processed with clean extractants (such as ultrapure water or supercritical carbon dioxide without modifiers) and diluted to volume without the use of chemicals.

(U) When solid samples are handled with acceptor phases that contaminate the solid matrix, the final volume is recorded as trash (also taking into account the likely dilution of the extract or digestate prior to the analysis) (e.g., Soxhlet extraction, microwave- or ultrasound-assisted extraction involving solvents other than water, supercritical fluid extraction involving modifiers)

(V) The solid residue that remains after solid samples are not fully dissolved or digested is likewise regarded as trash and must be added to the initial quantity (U).

Additional chemicals are not taken into account in the case of gaseous samples because they are addressed in the flow diagram's earlier sections. The total waste is then determined as follows: P + Q + R - S + T for liquids, P + Q + R - S + U + V for solids, and P + Q + R - S + R-S for gaseous samples.

The only exceptions are when the final volume contains the chemicals taken into account in R and S. In these cases, the total waste is determined as P + Q + T for liquid samples and P + Q + U + V for solid samples.

Principle 8: Number of analytes determined in a single run and samples analysed per hour⁸

Figure 8. Principle 8

Table 6: Number of Analytes analysed (per Hour)

Score	Analytes analysed (per Hour)
1	70
0.9	50
0.5	10
0	1

Table 5: Analytes Determined in a Single Run

Step	Single-Analyte Method	Multi-Analyte Method
Number of runs per sample	3 (one per drug)	1 (all three in one chromatographic run)
Time per run	15 minutes	20 minutes (total)
Analytes per hour	~12 (3 drugs × 4 runs)	~45 (3 drugs × 15 runs)
Reagent and solvent consumption	3× solvent per sample	1× solvent per sample
Instrument usage	3× longer	Efficient utilization

Score = 0.2429 × in (number of analytes determined in 1 h) - 0.0517

Principle 9: Select the most energy-intensive technique^10,11 used in the method and estimate the total power consumption of a single analysis in kWh

Figure 9(a). Principle 9(a)

· ≤ 0.1 kWh per sample: Score = 1 (Excellent, green method)

· 0.1–1.5 kWh per sample: Score = 0.5 (Moderate, acceptable)

· 1.5 kWh per sample: Score = 0 (Poor, non-green method)

Examples:

1. Ultrasound bath (130 W) operated for 0.75 h for 1 sample

2. Shaker agitator (50 W) operated for 18 h for 6 samples

Adjusted energy = Rated Power × Time × 0.4 (correction factor)

· It should be noted that a previous study that looked at the energy consumption of analytical equipment found that actual power levels were roughly 40% lower than the manufacturer's stated maximum value ^[12].

· For those systems that could not be physically measured, the authors of the same paper included a correction factor of 40%.

Energy per sample = Adjusted energy / Number of samples

Calculation:

1. Ultrasound Bath Energy Consumption:

· Rated Power = 130 W

· Time = 0.75 h

· Actual Power = 130 W × 0.4 = 52 W

· Energy per sample = 52 W × 0.75 h = 39 Wh/sample

2. Shaker Agitator Energy Consumption:

· Rated Power = 50 W

· Time = 18 h

· Actual Power = 50 W × 0.4 = 20 W

· Total Energy = 20 W × 18 h = 360 Wh

· Energy per sample = 360 Wh / 6 samples = 60 Wh/sample

Interpretation Based on Green Analytical Chemistry Criteria:

Energy Consumption Score (per sample):

· Ultrasound bath = 39 Wh → Mid-range performance → Score ≈ 0.8

· Shaker agitator = 60 Wh → Slightly above 50 Wh but well below 500 Wh → Score ≈ 0.75

Number of Sample Preparation Steps:

· Ultrasound method: 2 steps (e.g., weighing + extraction) → Score = 1

· Shaker method: 5 steps (e.g., weighing, pH adjustment, extraction, centrifugation, filtration) → Score ≈ 0.2

Defining a step in sample preparation is crucial at this stage. A step is defined as an operation or series of processes that results in a change in the sample matrix, including changes to its volume, characteristics, composition, phase separation, or even the analyte itself. Filtration, dilution, decantation, mineralization, extraction, centrifugation, sorption, analyte derivatization, drying, and lyophilization are a few typical sample preparation procedures ¹³.

Principle 10: Select the types of reagents used ¹⁴

Figure 10. Principle 10

Figure 11. Principle 11

No reagents or all bio-based reagents: Score = 1 (Green method)

· Some reagents bio-based: Score = 0.5 (Moderately green)

· No bio-based reagents: Score = 0 (non-green method)

Principle 11: Does the method involve the use of toxic reagents or solvents?¹⁵

For procedures which consume no toxic reagents, the score is 1.0, for others it is based on the following equation

Score = 0.156 × in (amount of reagent or solvent in g or mL) + 0.5898

Toxic towards inhalation, ingestion, dermal contact, or toxicity to aquatic life

Principle 12: Select the threats which are not avoided

Figure 12. Principle 12

Table 8: Number of unavoidable threats

No. of Threats	Score
Zero	1
1	0.8
2	0.6
3	0.4
4	0.2
>5	0

Table 9: References summary table of applied GAC in different analytical techniques

HPLC^{27, 28, 29}

HPTLC ⁴²

LC MS ^{43, 44}

GC ⁴⁵

UV ^{25, 35, 46, 47, 48, 49}

LC (Green Stationary Phases)⁵¹

IC⁵⁰^{, 53, 54, 59, 60}

RP-HPLC ^{20, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41}^{, 52, 55,
56, 57, 58, 61}

CONCLUSION:

AGREEprep represents a transformative step in bridging the gap between the theoretical ideals of Green Analytical Chemistry (GAC) and their practical implementation, particularly in the realm of sample preparation. By offering a standardized, intuitive, and scientifically robust framework for evaluating the greenness of analytical methods, AGREEprep not only enhances transparency but also empowers chemists to make informed, sustainable decisions at every stage of method development. Its visual scoring system and adaptability across diverse techniques foster a culture of continuous improvement, encouraging greener innovations without compromising analytical performance.

Looking ahead, the integration of AGREEprep into routine laboratory workflows, academic curricula, and regulatory frameworks holds immense potential. Coupling AGREEprep with artificial intelligence, automation, and digital lab platforms could pave the way for real-time green assessments and autonomous method optimization. As global emphasis on environmental sustainability intensifies, tools like AGREEprep will become indispensable not just as evaluative instruments, but as catalysts for a paradigm shift toward greener, cleaner, and more responsible analytical science. By embracing AGREEprep, the scientific community takes a decisive step toward a future where analytical excellence and environmental stewardship go hand in hand turning green chemistry principles from aspiration into actionable practice.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank B.K. Government Pharmacy College, Rajkot for their kind support during the review work for providing resources.

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Received on 30.01.2026 Revised on 10.03.2026

Accepted on 11.04.2026 Published on 27.05.2026

Available online from May 30, 2026

Asian J. Research Chem.2026; 19(3):199-210.

DOI: 10.52711/0974-4150.2026.00032

This work is licensed under a Creative Commons Attribution-Non Commercial-Share Alike 4.0 International License. Creative Commons License.

Positioning	Device Location	Sample Handling	Time Delay	Greenness Score	Example
Off-Line	Separate lab	Manual collection & transport	Hours to days	0.00	HPLC in QC lab analyzing tablets
At-Line	Near production line	Manual but immediate	Minutes	0.33	Portable NIR near granulator
On-Line	Connected to process line	Automated sampling	Seconds to minutes	0.66	On-line UV-Vis in reactor line
In-Line	Integrated inside process	No sample removal, direct	Real-time	1.00	NIR probe inside blender

Level of automation and miniaturization	Score
Automatic, miniaturized	1
Semi-automatic, miniaturized	0.75
Manual, miniaturized	0.5
Automatic, not miniaturized	0.5
Semi-automatic, not miniaturized	0.25
Manual, not miniaturized	0

Automation	Miniaturization	Greenness Score	Key Feature	Example
Automatic	Miniaturized	1	Fully automated, portable	Automated microfluidic API analysis
Semi-auto	Miniaturized	0.75	Partial automation, small scale	Semi-auto handheld Raman probe
Manual	Miniaturized	0.5	Manual operation, small scale	Manual microplate assay
Automatic	Not miniaturized	0.5	Automated but large equipment	Automated HPLC in QC lab
Semi-auto	Not miniaturized	0.25	Semi-auto with manual steps	Semi-auto UV-Vis requiring manual prep
Manual	Not miniaturized	0	Fully manual, large scale	Manual titration with traditional glassware

Name	Type	GAC Concern	Analytical Example (with Reference)
Acetonitrile^[16]	Solvent	Flammable, toxic to aquatic life, VOC emitter	Used as mobile phase in HPLC of paracetamol and caffeine¹⁷
Methanol	Solvent	Highly toxic, CNS depression, flammable	Sample prep for UV-Vis assay of metformin and glibenclamide ¹⁸
Chloroform	Solvent	Carcinogenic, ozone-depleting, volatile	Extraction of codeine phosphate from cough syrup in pharmaceutical analysis ¹⁹
Dichloromethane (DCM)	Solvent	Carcinogen suspect, high volatility, toxic to environment	Extraction of clobetasol propionate from creams for HPLC ²⁰
Tetrahydrofuran (THF)	Solvent	Peroxide-forming, flammable, respiratory irritant	HPLC analysis of polymer-drug conjugates in pharma formulations ²¹
n-Hexane	Solvent	Neurotoxic, flammable, environmental pollutant	Extraction of fatty acids in nutraceuticals using GC-MS ²²
Hydrazine	Reagent	Mutagenic, carcinogenic, explosive	Derivatization reagent for UV-spectrophotometric determination of isoniazid²³
Pyridine	Solvent/ Reagent	Toxic, flammable, bad odor, persistent	Used in derivatization for GC analysis of aspirin impurities²⁴
Trifluoroacetic acid (TFA)	Reagent/Modifier	Corrosive, bioaccumulative, toxic to aquatic life	Additive in HPLC for analyzing peptides and proteins²⁵
Phosphoric acid (conc.)	Acid/Base	Corrosive, hazardous waste management concern	Buffer in HPLC method for atenolol and amlodipine combination²⁶